title: Pyrethroid insecticides susceptibility of Stomoxys calcitrans (Linnaeus) and Stomoxys indicus Picard (Diptera: Muscidae) from cattle farms in southern Thailand

abstract: The susceptibility to six pyrethroid insecticides (permethrin, deltamethrin, alpha-cypermethrin, cypermethrin, lambda-cyhalothrin and bifenthrin), each at the recommended concentration, was evaluated for the two stable fly species Stomoxys calcitrans (Linnaeus) and Stomoxys indicus Picard, through tarsal contact using a World Health Organization (WHO) cone bioassay procedure. The field populations of stable flies were collected from three study sites (Songkhla, Phattalung and Satun provinces) in Thailand. The stable flies were exposed to insecticide-treated filter paper for 30 min and their knockdown counts at 30 min and 60 min and mortality counts at 12 hr and 24 hr were recorded. The S. calcitrans and S. indicus in Songkhla and Phattalung populations were moderately susceptible to pyrethroids for 24-hr mortality. Nonetheless, the Satun population of S. indicus was completely susceptible to permethrin with 100% mortality and the lowest susceptible to deltamethrin and bifenthrin. The results indicate a generally low susceptibility of stable flies to pyrethroids in the southern provinces of Thailand.

category: animal behavior and cognition


title: CameraEEG: Synchronized Recording of Video with Electroencephalogram data on an Android Application

abstract:
We present a novel android application named CameraEEG that enables synchronized acquisition of Electroencephalogram(EEG) and camera data using a smartphone. Audio-visual events of interest experienced by the subject were also recorded using a button press on the CameraEEG app. Unlike lab-restricted experiments, which usually constrain the subjects mobility, this wearable solution enables monitoring of the human brain during everyday life activities. The app was built using Android SDK version 28 and Smarting mobi SDK from mbraintrain. It works on all android devices having a minimum Android OS - Lollipop.

We successfully recorded thirty minutes of synchronized Video and EEG during eyes closed and walking tasks using the app. Event markers enabled by the subject using the app during walking tasks were also recorded. Timing tests showed that temporal synchronization of video and EEG data was good. We analysed the recorded data and were able to identify the task performed by the subject from the event markers. The power spectrum density of the two tasks showed different power spectrums with a peak in the alpha band for eyes closed task. We also provide android studio codes for download and detailed help documentation for the community to test the developed application.

category: bioengineering


title: Deep Learning-powered Bessel-beam Multi-parametric Photoacoustic Microscopy

abstract: Enabling simultaneous and high-resolution quantification of the total concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and cerebral blood flow (CBF), multi-parametric photoacoustic microscopy (PAM) has emerged as a promising tool for functional and metabolic imaging of the live mouse brain. However, due to the limited depth of focus imposed by the Gaussian-beam excitation, the quantitative measurements become inaccurate when the imaging object is out of focus. To address this problem, we have developed a hardware-software combined approach by integrating Bessel-beam excitation and conditional generative adversarial network (cGAN)-based deep learning. Side-by-side comparison of the new cGAN-powered Bessel-beam multi-parametric PAM against the conventional Gaussian-beam multi-parametric PAM shows that the new system enables high-resolution, quantitative imaging of CHb, sO2, and CBF over a depth range of [~]600 m in the live mouse brain, with errors 13-58 times lower than those of the conventional system. Better fulfilling the rigid requirement of light focusing for accurate hemodynamic measurements, the deep learning-powered Bessel-beam multi-parametric PAM may find applications in large-field functional recording across the uneven brain surface and beyond (e.g., tumor imaging).


title: Comprehensive Fitness Landscape of a Multi-Geometry Protein Capsid Informs Machine Learning Models of Assembly

abstract: Virus-like particles (VLPs) are non-infections viral-derived nanomaterials poised for biotechnological applications due to their well-defined, modular self-assembling architecture. Although progress has been made in understanding the complex effects that mutations may have on VLPs, nuanced understanding of the influence particle mutability has on quaternary structure has yet to be achieved. Here, we generate and compare the apparent fitness landscapes of two capsid geometries (T=3 and T=1 icosahedral) of the bacteriophage MS2 VLP. We find significant shifts in mutability at the symmetry interfaces of the T=1 capsid when compared to the wildtype T=3 assembly. Furthermore, we use the generated landscapes to benchmark the performance of in silico mutational scanning tools in capturing the effect of missense mutation on complex particle assembly. Finding that predicted stability effects correlated relatively poorly with assembly phenotype, we used a combination of de novo features in tandem with in silico results to train machine learning algorithms for the classification of variant effects on assembly. Our findings not only reveal ways that assembly geometry affects the mutable landscape of a self-assembled particle, but also establish a template for the generation of predictive mutational models of self-assembled capsids using minimal empirical training data.


title: Explainable autoencoder-based representation learning for gene expression data

abstract:
Modern machine learning methods have been extensively utilized in gene expression data analysis. In particular, autoencoders (AE) have been employed in processing noisy and heterogenous RNA-Seq data. However, AEs usually lead to "black-box" hidden variables difficult to interpret, hindering downstream experimental validation and clinical translation. To bridge the gap between complicated models and biological interpretations, we developed a tool, XAE4Exp (eXplainable AutoEncoder for Expression data), which integrates AE and SHapley Additive exPlanations (SHAP), a flagship technique in the field of eXplainable AI (XAI). It quantitatively evaluates the contributions of each gene to the hidden structure learned by an AE, substantially improving the expandability of AE outcomes. By applying XAE4Exp to The Cancer Genome Atlas (TCGA) breast cancer gene expression data, we identified genes that are not differentially expressed, and pathways in various cancer-related classes. This tool will enable researchers and practitioners to analyze high-dimensional expression data intuitively, paving the way towards broader uses of deep learning.

AvailabilityOpen source at https://github.com/QingrunZhangLab/Explainable-Deep-Autoencoder.

Contactsqingrun.zhang@ucalgary.ca and wliao@ucalgary.ca.

category: bioinformatics


title: Isolating salient variations of interest in single-cell transcriptomic data with contrastiveVI

abstract: Single-cell RNA sequencing (scRNA-seq) technologies enable a better understanding of previously unexplored biological diversity. Oftentimes, researchers are specifically interested in modeling the latent structures and variations enriched in one target scRNA-seq dataset as compared to another background dataset generated from sources of variation irrelevant to the task at hand. For example, we may wish to isolate factors of variation only present in measurements from patients with a given disease as opposed to those shared with data from healthy control subjects. Here we introduce Contrastive Variational Inference (contrastiveVI; https://github.com/suinleelab/contrastiveVI), a framework for end-to-end analysis of target scRNA-seq datasets that decomposes the variations into shared and target-specific factors of variation. On four target-background dataset pairs, we apply contrastiveVI to perform a number of standard analysis tasks, including visualization, clustering, and differential expression testing, and we consistently achieve results that agree with known biological ground truths.


title: Protein Complex Structure Prediction Powered by Multiple Sequence Alignment of Interologs from Multiple Taxonomic Ranks and AlphaFold2

abstract: AlphaFold2 is expected to be able to predict protein complex structures as long as a multiple sequence alignment (MSA) of the interologs of the target protein-protein interaction (PPI) can be provided. However, preparing the MSA of protein-protein interologs is a non-trivial task due to the existence of paralogs. In this study, a simplified phylogeny-based approach was applied to generate the MSA of interologs, which was then used as the input to AlphaFold2 for protein complex structure prediction. Extensively benchmarked this protocol on non-redundant PPI dataset including 107 bacterial PPIs and 442 eukaryotic PPIs, we show complex structures of 79.5% of the bacterial PPIs and 49.8% of the eukaryotic PPIs can be successfully predicted. Considering PPIs may not be conserved in species with long evolutionary distances, we further restricted interologs in the MSA to different taxonomic ranks of the species of the target PPI in protein complex structure prediction. We found the success rates can be increased to 87.9% for the bacterial PPIs and 56.3% for the eukaryotic PPIs if interologs in the MSA are restricted to a specific taxonomic rank of the species of each target PPI. Finally, we show the optimal taxonomic ranks for protein complex structure prediction can be selected with the application of the recalculated predicted TM-scores of the output models.


title: Protein Complex Structure Prediction Powered by Multiple Sequence Alignments of Interologs from Multiple Taxonomic Ranks and AlphaFold2


title: CSNet: Estimating cell-type-specific gene co-expression networks from bulk gene expression data

abstract: Inferring and characterizing gene co-expression networks has led to important insights on the molecular mechanisms of complex diseases. Most co-expression analyses to date have been performed on gene expression data collected from bulk tissues with different cell type compositions across samples. As a result, the co-expression estimates only offer an aggregate view of the underlying gene regulations and can be confounded by heterogeneity in cell type compositions, failing to reveal gene coordination that may be distinct across different cell types. In this paper, we describe a flexible framework for estimating cell-type-specific gene co-expression networks from bulk sample data, without making specific assumptions on the distributions of gene expression profiles in different cell types. We develop a novel sparse least squares estimator, referred to as CSNet, that is efficient to implement and has good theoretical properties. Using CSNet, we analyzed the bulk gene expression data from a cohort study on Alzheimers disease and identified previously unknown cell-type-specific co-expressions among Alzheimers disease risk genes, suggesting cell-type-specific disease pathology for Alzheimers disease.


title: Estimating cell-type-specific gene co-expression networks from bulk gene expression data with an application to Alzheimer's disease


title: A deep learning framework for predicting human essential genes from population and functional genomic data

abstract: Being able to predict essential genes intolerant to loss-of-function (LOF) mutations can dramatically improve our ability to identify genes associated with genetic disorders. Numerous computational methods have recently been developed to predict human essential genes from population genomic data; however, the existing methods have limited power in pinpointing short essential genes due to the sparsity of polymorphisms in the human genome. Here we present an evolution-based deep learning model, DeepLOF, which integrates population and functional genomic data to improve gene essentiality prediction. Compared to previous methods, DeepLOF shows unmatched performance in predicting ClinGen haploinsufficient genes, mouse essential genes, and essential genes in human cell lines. Furthermore, DeepLOF discovers 109 potentially essential genes that are too short to be identified by previous methods. Altogether, DeepLOF is a powerful computational method to aid in the discovery of essential genes.


title: Stabilized COre gene and Pathway Election uncovers pan-cancer shared pathways and a cancer specific driver

abstract:
Approaches systematically characterizing interactions via transcriptomic data usually follow two systems: (1) co-expression network analyses focusing on correlations between genes; (2) linear regressions (usually regularized) to select multiple genes jointly. Both suffer from the problem of stability: a slight change of parameterization or dataset could lead to dramatic alternations of outcomes. Here, we propose Stabilized Core gene and Pathway Election, or SCOPE, a tool integrating bootstrapped LASSO and co-expression analysis, leading to robust outcomes insensitive to variations in data. By applying SCOPE to six cancer expression datasets (BRCA, COAD, KIRC, LUAD, PRAD and THCA) in The Cancer Genome Atlas, we identified core genes capturing interaction effects in crucial pan-cancer pathways related to genome instability and DNA damage response. Moreover, we highlighted the pivotal role of CD63 as an oncogenic driver and a potential therapeutic target in kidney cancer. SCOPE enables stabilized investigations towards complex interactions using transcriptome data.

Availabilityhttps://github.com/QingrunZhangLab/SCOPE


title: Early exposure to UV radiation causes telomere shortening and poorer condition later in life

abstract:
Determining the contribution of elevated ultraviolet-B radiation (UVBR; 280 - 315 nm) to amphibian population declines is being hindered by a lack of knowledge about how different acute UVBR exposure regimes during early life history stages might affect post-metamorphic stages via long-term carryover effects. We acutely exposed tadpoles of the Australian green tree frog (Litoria caerulea) to a combination of different UVBR irradiances and doses in a multi-factorial experiment, and then reared them to metamorphosis in the absence of UVBR to assess carryover effects in subsequent juvenile frogs. Dose and irradiance of acute UVBR exposure influenced carryover effects into metamorphosis in somewhat opposing manners. Higher doses of UVBR exposure in larvae yielded improved rates of metamorphosis. However, exposure at a high irradiance resulted in frogs metamorphosing smaller in size and in poorer condition than frogs exposed to low and medium irradiance UVBR as larvae. We also demonstrate some of the first empirical evidence of UVBR-induced telomere shortening in vivo, which is one possible mechanism for life-history trade-offs impacting condition post-metamorphosis. These findings contribute to our understanding of how acute UVBR exposure regimes in early life affect later life-history stages, which has implications for how this stressor may shape population dynamics.

SUMMARY STATEMENTWe demonstrate physiological carryover effects in amphibians that link larval UV exposure to detrimental impacts on juvenile frogs, including telomere shortening, which has implications for how UV shapes amphibian populations.

category: physiology


title: The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion

abstract: Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Approximately 78% of cases are caused by mutations in the PKD1 gene, which encodes polycystin-1 (PC1). PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid residues of PC1 in a Pkd1-KO orthologous murine model of ADPKD dramatically suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase. This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.


title: The class I TCP transcription factor AtTCP8 is a modulator of phytohormone-responsive signaling networks

abstract:
The plant-specific TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR (TCP) transcription factor family is most closely associated with regulating plant developmental programs. Recently, TCPs were also shown to mediate host immune signaling, both as targets of pathogen virulence factors and regulators of plant defense genes. However, any comprehensive characterization of TCP gene targets is still lacking. Loss of the class I TCP AtTCP8 attenuates early immune signaling, and when combined with mutations in AtTCP14 and AtTCP15, additional layers of defense signaling in Arabidopsis thaliana. Here we focus on TCP8, the most poorly characterized of the three to date. We use chIP and RNA-sequencing to identify TCP8-bound gene promoters and differentially regulated genes in the tcp8 mutant, data sets that are heavily enriched in signaling components for multiple phytohormone pathways, including brassinosteroids (BRs), auxin, and jasmonic acid (JA). Using BR signaling as a representative example, we show that TCP8 directly binds and activates the promoters of the key BR transcriptional regulators BZR1 and BZR2/BES1. Furthermore, tcp8 mutant seedlings exhibit altered BR-responsive growth patterns and complementary reductions in BZR2 transcript levels, while the expressed protein demonstrates BR-responsive changes in subnuclear localization and transcriptional activity. We conclude that one explanation for the significant targeting of TCP8 alongside other TCP family members by pathogen effectors may lie in its role as a modulator of brassinosteroid and other plant hormone signaling pathways.

One Sentence SummaryOne member of a pathogen-targeted transcription factor family modulates phytohormone response networks and displays brassinosteroid-dependent cellular location and activity.

category: plant biology


title: CRISPR/Cas9 knockout of EPFL10 reduces stomatal density while maintaining photosynthesis and enhancing water conservation in rice

abstract:
Rice production is of paramount importance for global nutrition and potential yields will be detrimentally affected by climate change. Rice stomatal developmental genetics were explored as a mechanism to improve water use efficiency while maintaining yield under climate stress.

Gene-editing of STOMAGEN and its paralog, EPFL10, using CRISPR/Cas9 in rice cv. Nipponbare yielded lines with altered stomatal densities that were functionally characterized. CRISPR/Cas9 mediated knockouts of EPFL10 and STOMAGEN yielded lines with c. 80% and 25% of wild-type stomata, respectively.

epfl10 lines with small reductions in stomatal densities are able to conserve water to similar extents as stomagen lines with large stomatal density reductions but do not suffer from any concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation.

The duplicate of STOMAGEN, EPFL10, is a weak positive regulator of stomatal development in rice. epfl10 lines maintained wild-type physiological characteristics while conserving more water. Modest reductions in stomatal densities may be a climate-adaptive approach in rice that can safeguard yield.


title: The African mosquito-borne diseasosome: Geographical patterns and range expansion

abstract: Mosquito-borne diseases (MBDs) such as malaria, dengue, and Rift Valley fever threaten public health and food security globally. Despite their cohesive nature, they are typically treated as distinct entities. Applying biological system analysis to the African MBDs from a One Health perspective, we provide the first biogeographic description of the African mosquito fauna corresponding with the pathogens they transmit. After compiling records accumulated over a century, we find that there are 677 mosquito species in Africa, representing 16 genera, and 151 mosquito-borne pathogens (MBPs) circulating primarily among wild tetrapods, dominated by viruses (95) and protozoans (47). We estimate that reported MBPs represent [~]1% of the actual number. Unlike mosquitoes, African arboviruses and mammalian plasmodia represent a higher share of the Worlds total based on the area - species richness relationship (P<0.0001), explaining the disproportional large share of global MBPs that originated from Africa. Species richness of African mosquitoes and MBPs are similarly concentrated along the equator, peaking in central Africa, with a secondary "ridge" along eastern Africa. Moderate diversity and low endemicity in mosquitoes across the Sahel reveals a fauna with high propensity for long-range migration. Regional differences in species richness, endemicity, and composition agreed with country-based results. The composition of mosquitoes and MBPs separates sub-Saharan Africa from north Africa, in accordance with the Palearctic and Afrotropical faunal realms, and west and central Africa are clustered together distinctly from the cluster of eastern and southern Africa. With [~]25% of the species occupying a single country, [~]50% in 1-3 countries and <5% found in >25 countries, the typical ranges of both mosquitoes and MBPs are surprisingly small. The striking similarity in diversity and especially in range distributions of mosquitoes and MBPs suggest that most MBPs are transmitted by one or few narrow-range mosquito vectors. Exceptionally widespread mosquito species (e.g., Ae. aegypti, Cx. quinquefasciatus, and 10 Anopheles species) feed preferentially on people and domestic animals, and nearly half are windborne migrants. Likewise, exceptionally widespread MBPs are transmitted between people or domestic animals and are vectored by one or more of the aforementioned widespread mosquitoes. Our results suggest that few MBPs have undergone a dramatic range expansion, after adapting to people or domestic animals as well as to exceptionally-widespread mosquitoes. During the intermediate phase of range expansion, MBPs extend their vector and vertebrate host ranges with a concomitant gradual increase in geographical range. Because range size may serve as a marker of the phase of range expansion, ranking the African MBPs according to range, we identified several MBPs that pose elevated risk for disease emergence (e.g., Wesselsbron virus). Taken together, our database, approach, and results can help improve MBD surveillance and lead to a better understanding of disease emergence. This knowledge has the potential to improve capacity to prevent and mitigate new and emerging MBD threats.

category: zoology


title: How synaptic strength, short-term plasticity, and temporal factors contribute to neuronal spike output

abstract: To compute spiking responses, neurons integrate inputs from thousands of synapses whose strengths span an order of magnitude. Intriguingly, in mouse neocortex, the small minority of  strong synapses is found predominantly between similarly tuned cells, suggesting they are the synapses that determine a neurons spike output. This raises the question of how other computational primitives, such as  background activity from the majority of synapses, which are  weak, short-term plasticity, and temporal synchrony contribute to spiking. First, we combined extracellular stimulation and whole-cell recordings in mouse barrel cortex to map the distribution of excitatory postsynaptic potential (EPSP) amplitudes and paired-pulse ratios of excitatory synaptic connections converging onto individual layer 2/3 (L2/3) neurons. While generally net short-term plasticity was weak, connections with EPSPs > 2 mV displayed pronounced paired-pulse depression. EPSP amplitudes and paired-pulse ratios of connections converging onto the same neurons spanned the full range observed across L2/3 and there was no indication that strong synapses nor those with particular short-term plasticity properties were associated with particular cells, which critically constrains theoretical models of cortical filtering. To investigate how different computational primitives of synaptic information processing interact to shape spiking, we developed a computational model of a pyramidal neuron in the rodent L2/3 circuitry: firing rates and pairwise correlations of presynaptic inputs were constrained by in vivo observations, while synaptic strength and short-term plasticity were set based on our experimental data. Importantly, we found that the ability of strong inputs to evoke spiking critically depended on their high temporal synchrony and high firing rates observed in vivo and on synaptic background activity - and not primarily on synaptic strength, which in turn further enhanced information transfer. Depression of strong synapses was critical for maintaining a neurons responsivity and prevented runaway excitation. Our results provide a holistic framework of how cortical neurons exploit complex synergies between temporal coding, synaptic properties, and noise in order to transform synaptic inputs into output firing.

category: neuroscience


title: Proprioceptive Re-alignment drives Implicit Sensorimotor Adaptation

abstract: Multiple learning processes contribute to successful goal-directed actions in the face of changing physiological states, biomechanical constraints, and environmental contexts. Amongst these processes, implicit sensorimotor adaptation is of primary importance, ensuring that movements remain well-calibrated and accurate. A large body of work on reaching movements has emphasized how adaptation centers on an iterative process designed to minimize visual errors. The role of proprioception has been largely neglected, thought to play a passive role in which proprioception is affected by the visual error but does not directly contribute to adaptation. Here we present an alternative to this visuo-centric framework, arguing that that implicit adaptation can be understood as minimizing a proprioceptive error, the distance between the perceived hand position and its intended goal. We use this proprioceptive re-alignment model (PReMo) to re-examine many phenomena that have previously been interpreted in terms of learning from visual errors, as well as offer novel accounts for unexplained phenomena. We discuss potential challenges for this new perspective on implicit adaptation and outline a set of predictions for future experimentation.


title: Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

abstract: Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.


title: Directed motor actions and choice signalling drive cortical acetylcholine dynamics.

abstract: Numerous cognitive functions including attention, learning, and plasticity are influenced by the dynamic patterns of acetylcholine release across the brain. How acetylcholine mediates these functions in cortex remains unclear, as the spatiotemporal relationship between cortical acetylcholine and behavioral events has not been precisely measured across task learning. To dissect this relationship, we quantified motor behavior and sub-second acetylcholine dynamics in primary somatosensory cortex during acquisition and performance of a tactile-guided object localization task. We found that acetylcholine dynamics were spatially homogenous and directly attributable to whisker motion and licking, rather than sensory cues or reward delivery. As task performance improved across training, acetylcholine release to the first lick in a trial became dramatically and specifically potentiated, paralleling the emergence of a choice-signalling basis for this motor action. These results show that acetylcholine dynamics in sensory cortex are driven by directed motor actions to gather information and act upon it.


title: The role of the angular gyrus in semantic cognition - A synthesis of five functional neuroimaging studies

abstract: Semantic knowledge is central to human cognition. The angular gyrus (AG) is widely considered a key brain region for semantic cognition. However, the role of the AG in semantic processing is controversial. Key controversies concern response polarity (activation vs. deactivation) and its relation to task difficulty, lateralization (left vs. right AG), and functional-anatomical subdivision (PGa vs. PGp subregions). Here, we combined the fMRI data of five studies on semantic processing (n = 172) and analyzed the response profiles from the same anatomical regions-of-interest for left and right PGa and PGp. We found that the AG was consistently deactivated during non-semantic conditions, whereas response polarity during semantic conditions was inconsistent. However, the AG consistently showed relative response differences between semantic and non-semantic conditions, and between different semantic conditions. A combined analysis across all studies revealed that AG responses could be best explained by separable effects of task difficulty and semantic processing demand. Task difficulty effects were stronger in PGa than PGp, regardless of hemisphere. Semantic effects were stronger in left than right AG, regardless of subregion. These results suggest that the AG is engaged in both domain-general task-difficulty-related processes and domain-specific semantic processes. In semantic processing, we propose that left AG acts as a "multimodal convergence zone" that binds different semantic features associated with the same concept, enabling efficient access to task-relevant features.


title: A phase-shifting anterior-posterior network organizes global phase relations

abstract: Prior research has identified a variety of task-dependent networks that form through inter-regional phase-locking of oscillatory activity as neural correlates of specific behaviors. Despite ample knowledge of task-specific functional networks, general rules governing global phase relations have not been investigated. In order to discover such general rules, we focused on phase modularity, measured as the degree to which global phase relations in EEG comprised distinct synchronized clusters interacting with one another at large phase lags. Synchronized clusters were detected with a standard community-detection algorithm, and the level of phase modularity was quantified by the index q. Our findings suggest that phase modularity is functionally consequential since (1) temporal distribution of q was invariant across a broad range of frequencies (3-50 Hz examined) and behavioral conditions (resting with the eyes closed or watching a silent nature video), and (2) neural interactions (measured as power correlations) in beta-to-gamma bands consistently increased in high-modularity states. Notably, we found that the mechanism controlling phase modularity is remarkably simple. A network comprising anterior-posterior long-distance connectivity coherently shifted phase relations from low-angles (|{Delta}{theta}| < {pi}/4) in low-modularity states (bottom 5% in q) to high-angles (|{Delta}{theta}| > 3{pi}/4) in high-modularity states (top 5% in q), accounting for fluctuations in phase modularity. This anterior-posterior network likely plays a fundamental functional role as it controls phase modularity across a broad range of frequencies and behavioral conditions. These results may motivate future investigations into the functional roles of phase modularity as well as the anterior-posterior network that controls it.


title: The role of intraspinal sensory neurons in the control of quadrupedal locomotion

abstract: From swimming to walking and flying, animals have evolved specific locomotor strategies to thrive in different habitats. All types of locomotion depend on integration of motor commands and sensory information to generate precise movements. Cerebrospinal fluid-contacting neurons (CSF-cN) constitute a vertebrate sensory system that monitors CSF composition and flow. In fish, CSF-cN modulate swimming activity in response to changes in pH and bending of the spinal cord, yet their role in higher vertebrates remains unknown. We used mouse genetics to study their function in quadrupedal locomotion and found that CSF-cN are directly integrated into spinal motor circuits by forming connections with motor neurons and premotor interneurons. Elimination of CSF-cN selectively perturbs the accuracy of foot placement required for skilled movements at the balance beam and horizontal ladder. These results identify an important role for mouse CSF-cN in adaptive motor control and indicate that this sensory system evolved a novel function from lower vertebrates to accommodate the biomechanical requirements of terrestrial locomotion.


title: CSF1R inhibition by small molecule affects T-helper cell differentiation independently of microglia depletion

abstract:
Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissueresident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only, but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation. In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data.

Significance statementHere we show that CSF1R inhibition affects not only innate immune cells and microglia, but also the adaptive immune compartment by suppressing Th1/Th2 differentiation of CD4+ cells, independently of microglia depletion.

category: immunology


title: Novel regimens of bedaquiline-pyrazinamide combined with moxifloxacin, rifabutin, delamanid and/or OPC-167832 in murine tuberculosis models

abstract: A recent landmark trial showed a 4-month regimen of rifapentine, pyrazinamide, moxifloxacin and isoniazid (PZMH) to be non-inferior to the 6-month standard of care. Here, two murine models of tuberculosis were used to test whether novel regimens replacing rifapentine and isoniazid with bedaquiline and another drug would maintain or increase the sterilizing activity of the regimen. In BALB/c mice, replacing rifapentine in the PZM backbone with bedaquiline (i.e., BZM) significantly reduced both lung CFU counts after 1 month and the proportion of mice relapsing within 3 months after completing 1.5 months of treatment. Addition of rifabutin to BZM (BZMRb) further increased the sterilizing activity. In the C3HeB/FeJ mouse model characterized by caseating lung lesions, treatment with BZMRb resulted in significantly fewer relapses than PZMH after 2 months of treatment. A regimen combining the new DprE1 inhibitor OPC-167832 and delamanid (BZOD) also had superior bactericidal and sterilizing activity compared to PZM in BALB/c mice and was similar in efficacy to PZMH in C3HeB/FeJ mice. Thus, BZM represents a promising backbone for treatment-shortening regimens. Given the prohibitive drug-drug interactions between bedaquiline and rifampin or rifapentine, the BZMRb regimen represents the best opportunity to combine, in one regimen, the treatment-shortening potential of the rifamycin class with that of BZM and deserves high priority for evaluation in clinical trials. Other 4-drug BZM-based regimens and BZOD represent promising opportunities for extending the spectrum of treatment-shortening regimens to rifamycin- and fluoroquinolone-resistant tuberculosis.

category: microbiology


title: The microRNA miR-132 is a key regulator of lymphatic vascular remodelling

abstract: Lymphangiogenesis (growth of new lymphatic vessels), and lymphatic remodelling more broadly, are important for disease progression in cancer, lymphedema and the pulmonary disease lymphangioleiomyomatosis. Multiple molecular pathways which signal for aspects of lymphangiogenesis are known but little is understood about their co-ordinate regulation in lymphatic endothelial cells (LECs). Small RNA molecules co-ordinately regulate complex biological processes, but knowledge about their involvement in lymphangiogenesis is limited. Here we used high-throughput small RNA sequencing of LECs to identify microRNAs (miRs) regulating lymphatic remodelling driven by the lymphangiogenic growth factors VEGF-C and VEGF-D. We identified miR-132 as up-regulated by both growth factors, and demonstrated that inhibiting miR-132 in LECs in vitro blocked cell proliferation and tube formation, key steps in lymphangiogenesis. We showed that miR-132 is expressed in human LECs in vivo in the lymphatics of human breast tumours expressing VEGF-D. Importantly, we demonstrated that inhibiting miR-132 in vivo blocked many aspects of lymphangiogenesis in mice. Finally, we identified mRNAs regulated by miR-132 in LECs, by sequencing after RNA-protein cross-linking and Argonaute immunoprecipitation, which demonstrated how miR-132 co-ordinately regulates signalling pathways in lymphangiogenesis. This study shows miR-132 is a critical regulator of lymphangiogenesis and a potential target for therapeutically manipulating lymphatic remodelling in disease.

category: cell biology


title: Accumulation of TERT in Mitochondria Shows Two Opposing Effects on Apoptosis

abstract: Telomerase reverse transcriptase (TERT) is a protein that catalyzes the reverse transcription of telomere elongation. TERT is also expected to play a noncanonical role beyond telomere lengthening since it localizes not only in the nucleus but also in mitochondria, where telomeres do not exist. Several studies have reported that mitochondrial TERT regulates apoptosis induced by oxidative stress. However, there remains controversy about whether mitochondrial TERT promotes or inhibits apoptosis, mainly due to the lack of information on changes in the TERT distribution in individual cells over time. Here we simultaneously detected apoptosis and TERT localization after oxidative stress in individual HeLa cells by live-cell tracking. This tracking revealed that the stress-induced accumulation of TERT in mitochondria caused apoptosis but that the accumulation positively correlated with the time until cell death. The results suggest a new model in which mitochondrial TERT has two opposing effects at different stages of apoptosis: it predetermines apoptosis at the first stage of cell-fate determination but also delays apoptosis at the second stage. Because these distinct effects respectively support both sides of the controversy regarding the role of mitochondrial TERT in apoptosis, our model integrates two opposing hypotheses. Furthermore, detailed statistical analysis of TERT mutations, which have been predicted to inhibit TERT transport to mitochondria, revealed that these mutations suppress apoptosis independent of the mitochondrial localization of TERT. Together, these results indicate that the non-canonical functions of TERT affect a wide range of pathways.


title: Coagulation Factor IIIa (f3a) Knockdown in Zebrafish Leads to Defective Angiogenesis and Mild Bleeding Phenotype

abstract:
AimTissue factor (TF), an initiator of the extrinsic coagulation pathway, is crucial for embryogenesis, as mice lacking TF are embryonically lethal (E10.5). This lethality may be attributed to defects in vascular development and circulatory failure, suggesting additional roles for TF in embryonic development beyond coagulation. In this study, we characterized the role of one of the TF paralogs (f3a) using a zebrafish model.

MethodsTo understand the TF evolution across different species, we performed molecular phylogenetic and sequence homology analysis. The expression of f3a during embryonic developmental stages was determined by RT-PCR. Endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) was used to image the vascular development. The role of f3a during embryonic development was investigated by mRNA knockdown using Morpholinos (MO), an antisense-based oligonucleotide strategy. The f3a morphants were examined at 52 hpf for defects in morphological appearance, bleeding, and vascular patterning.

ResultsSpatiotemporal expression of f3a by qPCR revealed expression in all developmental stages, suggesting that f3a transcripts are both maternally and zygotically expressed. High expression of f3a from 28 hpf to 36 hpf confirmed the role of in the development of the yolk sac, circulation, and fins. f3a MO-injected embryos showed morphological abnormalities, including shorter body lengths and crooked tails. O-dianisidine staining showed f3a MO-injected embryos exhibited bleeding in the trunk (5.44%) and head (9.52%) regions. Using endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX), imaging of caudal vein plexus, which forms immediately following the onset of circulation and sprouting, showed a 3-fold decrease in f3a morphants versus controls at 48 hpf, suggesting a potential role for f3a in flow-induced angiogenesis.

Conclusionf3a is essential for angiogenesis, in addition to its involvement in hemostasis.

Key PointsO_LIf3a knockdown by antisense morpholino oligonucleotide (MO)-approach shows bleeding phenotype.
C_LIO_LIf3a is essential for angiogenesis, in addition to its involvement in hemostasis.
C_LI

category: developmental biology


title: Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

abstract: The GM2 gangliosidoses Tay-Sachs disease and Sandhoff disease (SD) are respectively caused by mutations in the HEXA and HEXB genes encoding the  and {beta} subunits of {beta}-N-acetylhexosaminidase (Hex). The consequential accumulation of ganglioside in the brain leads to severe and progressive neurological impairment. There are currently no approved therapies to counteract or reverse the effects of GM2 gangliosidosis. Adeno-associated vector (AAV)-based investigational gene therapy (GT) products have raised expectations but come with safety and efficacy issues that need to be addressed. Thus, there is an urgent need to develop novel therapies targeting the CNS and other affected tissues that are appropriately timed to ensure pervasive metabolic correction and counteract disease progression. In this report, we show that the sequential administration of lentiviral vector (LV)-mediated intracerebral (IC) GT and bone marrow transplantation (BMT) in pre-symptomatic SD mice provide a timely and long-lasting source of the Hex enzyme in the central and peripheral nervous systems and peripheral tissues, leading to global rescue of the disease phenotype. Combined therapy showed a clear therapeutic advantage compared to individual treatments in terms of lifespan extension and normalization of the neuroinflammatory and neurodegenerative phenotypes of the SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in the brain tissues that single treatments failed to achieve. Our results highlight the complementary and synergic mode of action of LV-mediated IC GT and BMT, clarify the relative contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of enzymatic activity that is required to achieve a significant therapeutic benefit, with important implications for the monitoring and interpretation of ongoing experimental therapies, and for the design of more effective treatment strategies for GM2 gangliosidosis.

category: genetics


title: Meta analysis of variant predictions in congenital adrenal hyperplasia caused by mutations in CYP21A2.

abstract:
ContextCYP21A2 deficiency represents 95% of congenital adrenal hyperplasia cases (CAH), a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provides critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction.

ObjectiveAnalyze the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of the CYP21A2.

MethodsSNVs of the CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of the CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors.

ResultsSNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD).

ConclusionFrom our analysis, we identified four predictors of CYP21A2 pathogenicity with good performance. These results can be used for future analysis to infer the impact of uncharacterized SNVs in CYP21A2.


title: The Biomechanics of the Locust Ovipositor Valves: a Unique Digging Apparatus

abstract:
The female locust has a unique mechanism for digging in order to deposit its eggs deep in the ground. It utilizes two pairs of sclerotized valves to displace the granular matter, while extending its abdomen as it propagates underground. This ensures optimal conditions for the eggs to incubate, and provides them with protection from predators. Here, two major axes of operation of the digging valves are identified, one in parallel to the propagation direction of the ovipositor, and one perpendicular to it. The direction-dependent biomechanics of the locust major, dorsal digging valves are quantified and analyzed, under forces in the physiological range and beyond, considering hydration level, as well as the females age, or sexual maturation state. Our findings reveal that the responses of the valves to compression forces in the specific directions change upon sexual maturation to follow their function, and depend on environmental conditions. Namely, in the physiological force range, the valves are resistant to mechanical failure. In addition, mature females, which lay eggs, have stiffer valves, up to roughly nineteen times the stiffness of the pre-mature locusts. The valves are stiffer in the major working direction, corresponding to soil shuffling and compression, compared to the direction of propagation. Hydration of the valves reduces their stiffness but increases their resilience against failure. These findings provide mechanical and materials guidelines for the design of novel non-drilling excavating tools, including 3D-printed anisotropic materials based on composites.

Statement of significanceThe female locust lay its eggs underground in order to protect them from predators and to provide them with optimal conditions for hatching. In order to dig into the ground, it uses two pairs of valves: The ventral pair is plugged as a wedge, while the dorsal pair performs the digging of the oviposition tunnel. We study the mechanical response of the digging valves, depending on age, hydration level and direction of operation. Our findings show that during the course of roughly two weeks in the life of the adult female, the digging valves become up to nineteen-fold stiffer against failure, in order to fulfill their function as diggers. While hydration reduces the stiffness, it also increases the resilience against failure and renders the valves unbreakable within the estimated physiological force range and beyond. The digging valves are consistently stiffer in the digging direction than in the perpendicular direction, implying on their form-follows-function design.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: EasyFlow: User-friendly Workflow for Image-based Droplet Analysis with Multipurpose Modules

abstract: Droplet-based experimental platforms allow researchers to perform massive parallelization and high-throughput studies, such as single-cell experiments. Even though there are various options of image analysis software to evaluate the experiment, selecting the right tools require experience and is time consuming. Experts and sophisticated workflow are required to perform the analysis, especially to detect the droplets and analyze their content. There is need for user-friendly droplet analysis pipelines that can be adapted in laboratories with minimum learning curve. Here, we provide a user-friendly workflow for image-based droplet analysis. The workflow comprises of a) CellProfiler-based image-analysis pipeline and b) accompanied with web application that simplifies the analysis and visualization of the droplet-based experiment. We construct necessary modules in CellProfiler (CP) to detect droplets and export the results into our web application. Using the web application, we are able to process and provide basic profiles of the droplet experiment (droplet sizes, droplet signals, sizes-signals plot, and strip plot for each label/condition). We also add a specific module for growth heterogeneity studies in bacteria populations that includes single cell viability analysis and probability distribution of minimum inhibition concentration (MIC) values in population. Our pipeline is usable for both poly- and monodisperse droplet emulsions.


title: Comparative oncology reveals DNMT3B as a therapeutic vulnerability in soft-tissue sarcoma

abstract: Undifferentiated pleomorphic sarcoma (UPS), an aggressive subtype of soft-tissue sarcoma (STS), is exceedingly rare in humans and lacks effective, well-tolerated therapies. In contrast, STS are relatively common in canine companion animals; thus, incorporation of veterinary patients into studies of UPS offers an exciting opportunity to develop novel therapeutic strategies for this rare human disease. Genome-wide studies have demonstrated that UPS is characterized by aberrant patterns of DNA methylation. However, the mechanisms and impact of this epigenetic modification on UPS biology and clinical behavior are poorly understood. Leveraging cell lines and tissue specimens derived from human and canine patients, we discovered that the DNA methyltransferase DNMT3B is overexpressed in UPS relative to normal mesenchymal tissues and associated with a poor prognosis. Consistent with these findings, genetic DNMT3B depletion strongly inhibited UPS cell proliferation and tumor progression. However, existing hypomethylating agents, including the clinically approved drug 5-aza-2-deoxycytidine and the DNMT3B-inhibiting tool compound nanaomycin A, were ineffective in UPS due to cellular uptake and toxicity issues. Thus, further development of DNMT3B-targeting strategies for these patients is critical.

category: cancer biology


title: An enzyme-based system for the extraction of small extracellular vesicles from plants

abstract: Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are considered to be the next generation of nanocarrier platforms for biotherapeutics and drug delivery. However, EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitation of isolation methods, the extraction efficiency is low, resulting in the waste of a large number of plants, especially rare and expensive medicinal plants.There are few studies comparing EVs and NVs. To overcome these challenges, we proposed and validated a novel method for the isolation of plant EVs by degrading the plant cell wall with enzymes to release the EVs in the cell wall, making it easier for EVs to break the cell wall barrier and be collected. We extracted EVs from the roots of Morinda officinalis by enzymatic degradation(MOEVs) and nanoparticles by grinding method (MONVs) as a comparison group. The results showed smaller diameter and higher yield of MOEVs.Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxicity and promoted the expression of miR-155. The difference is that the promotion of miR-155 by MOEVs is dose-effective. More importantly, MOEVs and MONVs are naturally characterized by bone enrichment. These results support that EVs in plants can be efficiently extracted by enzymatic cell wall digestion and also confirm the potential of MOEVs as therapeutic agents and drug carriers.

category: pharmacology and toxicology


title: Live-cell microscopy or fluorescence anisotropy with budded baculoviruses - which way to go with measuring ligand binding to M4 muscarinic receptors?

abstract:
M4 muscarinic receptor is a G protein-coupled receptor that has been associated with alcohol and cocaine abuse, Alzheimers disease and schizophrenia which makes it an interesting drug target. For many G protein-coupled receptors, the development of high-affinity fluorescence ligands has expanded the options for high throughput screening of drug candidates and serve as useful tools in fundamental receptor research. So far, the lack of suitable fluorescence ligands has limited studying M4 receptor ligand binding. Here, we explored the possibilities of using fluorescence-based methods for studying binding affinity and kinetics to M4 receptor of both labeled and unlabeled ligands. We used two TAMRA-labeled fluorescence ligands, UR-MK342 and UR-CG072, for assay development. Using budded baculovirus particles as M4 receptor preparation and fluorescence anisotropy method, we determined the affinities and binding kinetics of both fluorescence ligands. The fluorescence ligands could also be used as reported probes for determining binding affinities of a set of unlabeled ligands. Based on these results, we took a step further towards a more natural signaling system and developed a method using live CHO-K1-hM4R cells and automated fluorescence microscopy suitable for routine determination of unlabeled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both developed methods were suitable for measuring fluorescence ligand saturation binding, association and dissociation kinetics as well as for screening binding affinities of unlabeled ligands.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/473643v1_ufig1.gif" ALT="Figure 1">
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title: Natural variation in the control of flowering and shoot architecture in diploid Fragaria species

abstract: In perennial fruit and berry crops of the Rosaceae family, flower initiation occurs in late summer or autumn after downregulation of a strong repressor TERMINAL FLOWER1 (TFL1) and flowering and fruiting takes place the following growing season. Rosaceous fruit trees typically form two types of axillary shoots, short flower-bearing shoots called spurs and long shoots that are respectively analogous to branch crowns and stolons in strawberry. However, regulation of flowering and shoot architecture differs between species and environmental and endogenous controlling mechanisms have just started to emerge. In woodland strawberry (Fragaria vesca L.), long days maintain vegetative meristems and promote stolon formation by activating TFL1 and GIBBERELLIN 20-OXIDASE4 (GA20ox4), respectively, while silencing of these factors by short days and cool temperatures induces flowering and branch crown formation. We characterized flowering responses of 14 accessions of seven diploid Fragaria species native to diverse habitats in the northern hemisphere, and selected two species with contrasting environmental responses, F. bucharica Losinsk. and F. nilgerrensis Schlecht. ex J. Gay for detailed studies together with F. vesca. Similar to F. vesca, F. bucharica was induced to flower in short days at 18{degrees}C and regardless of photoperiod at 11{degrees}C after silencing of TFL1. F. nilgerrensis maintained higher TFL1 expression level and likely required cooler temperatures or longer exposure to inductive treatments to flower. We also found that high expression of GA20ox4 was associated with stolon formation in all three species, and its downregulation by short days and cool temperature caused branch crown formation in F. vesca and F. nilgerrensis, although the latter did not flower. F. bucharica, in contrast, rarely formed branch crowns, regardless of flowering or GA20ox4 expression level. Our findings highlighted diploid Fragaria species as a rich source of genetic variation controlling flowering and plant architecture, with potential applications in breeding of Rosaceous crops.


title: Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria

abstract: Mutations in FBXO7 have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7 ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each others functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7 E3 ubiquitin ligase.


title: Neuro-anatomical and neuro-molecular markers in salience network and their integration in machine learning-based prediction of cognitive dysfunction in mild cognitive impairment

abstract: Recent studies indicate disrupted functional mechanisms of salience network regions, especially right anterior insula (RAI), left AI (LAI), and anterior cingulate cortex (ACC), in mild cognitive impairment (MCI). However, the underlying neuro-anatomical and neuro-molecular mechanisms in these regions are not clearly understood yet. It is also unknown whether integration of multi-modal neuro-anatomical and neuro-molecular markers could predict cognitive impairment better in MCI. Herein we quantified neuro-anatomical volumetric markers via structural magnetic resonance imaging (sMRI) and neuro-molecular amyloid markers via positron emission tomography with Pittsburgh compound B (PET PiB) in SN regions of MCI (n = 33) and healthy controls (n = 27). We found that neuro-anatomical markers are significantly reduced while neuro-molecular markers are significantly elevated in SN nodes of MCI compared to healthy controls (p < 0.05). These altered markers in MCI patients were associated with their worse cognitive performance (p < 0.05). Our machine learning-based modeling further suggested that the integration of multi-modal markers predicts cognitive impairment in MCI superiorly compared to using single modality-specific markers. Overall, these findings shed light on the underlying neuro-anatomical volumetric and neuro-molecular amyloid alterations in SN regions and show the significance of multi-modal markers integration approach in better predicting cognitive impairment in MCI.


title: What do zebrafish prefer? Directional and color preferences in maze tasks

abstract: Studies regarding the animals innate preferences help elucidate and avoid probable sources of bias and serve as a reference to improve and develop new behavioral tasks. In zebrafish research, the results of innate directional and color preferences are often not replicated between research groups or even inside the same laboratory raising huge concerns on the replicability and reproducibility. Thus, this study aimed to investigate the male and female zebrafish innate directional and color preferences in the plus-maze and T-maze behavioral tasks. As revealed by the percentage of time spent in each zone of the maze, our results showed that males and females zebrafish demonstrated no difference in directional preference in the plus-maze task. Surprisingly, male and female zebrafish showed color preference differences in the plus-maze task; males did not show any color preference, while female zebrafish demonstrated a red preference compared to white, blue, and yellow colors. Moreover, both male and female zebrafish demonstrated a strong black color preference compared to the white color in the T-maze task. Thus, our results demonstrate the importance of innate preference assays involved with the directionality of the apparatus or the application of colors as a screening process conducting behavioral tests (e.g., anxiety, learning and memory assessment, locomotion, and preference) and highlight the need to analyze sex differences.


title: Optineurin deficiency induces patchy hair loss but it is not sufficient to cause amyotrophic lateral sclerosis in mice

abstract: Genetic alterations and environmental factors contribute towards pathogenesis of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. Various types of mutations in the autophagy receptor protein optineurin (coded by OPTN gene) including deletions are causatively associated with ALS. To explore the role of OPTN in ALS pathogenesis, we used Optn knockout mice to study the features of ALS. The Optn-deficient mice did not show kyphosis, loss of body weight, weakening of front paw-grip strength or limb muscle strength. However, several Optn-deficient mice showed patchy loss of hair, which increased with age. Our results suggest that optineurin deficiency alone is not sufficient to induce ALS-like symptoms in mice. We suggest that optineurin deficiency may require cooperation with other genetic or environmental factors to cause ALS. Since endoplasmic reticulum (ER) stress plays an important role in ALS pathogenesis, and Optn modulates ER stress response signaling, Optn deficiency may contribute to ALS pathogenesis partly by potentiating ER stress response signaling.


title: Single-cell RNA-seq analysis reveals lung epithelial cell-specific contributions of Tet1 to allergic inflammation

abstract: Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. In order to explore the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation, we established a model of HDM-induced lung inflammation in Tet1 knockout and littermate wildtype mice and studied EpCAM+ lung epithelial cells using single-cell RNA-seq analysis. We identified eight EpCAM+ lung epithelial cell types, among which AT2 cells were the most abundant. HDM challenge increased the percentage of alveolar progenitor cells (AP), broncho alveolar stem cells (BAS), and goblet cells, and decreased the percentage of AT2 and ciliated cells. Bulk and cell-type-specific analysis identified genes subject to Tet1 regulation and linked to augmented lung inflammation, including alarms, detoxification enzymes, oxidative stress response genes, and genes in tissue repair. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Collectively, our data showed that lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, with an overall effect of promoting allergen-induced lung inflammation.


title: ANTIBODY-INDEPENDENT ANTITUMOR EFFECTS OF CD32A-CHIMERIC RECEPTOR T CELLS: IMPLICATIONS FOR BREAST CANCER PROGNOSIS AND TREATMENT.

abstract: Fc{gamma} RIIA (CD32A) and their ligands, including the immunoglobulin Fc fragment and pentraxins, are key players in a variety of innate immune responses. Still unclear is whether additional ligands of CD32A do exist. The objective of this study is to demonstrate that CD32A-chimeric receptor (CR) can be utilized for the identification of CD32A cell surface ligand(s). Among fifteen cancer cell lines tested, CD32A-CR T cells recognized three of breast cancer (BC) including the MDA-MB-468 and one colorectal carcinoma (HT29) in the absence of targeting antibodies. Conjugation of sensitive BC cells with CD32A-CR T cells induced CD32A polarization and down-regulation, CD107 release, and mutual cell elimination in vitro. Conversely, normal fibroblasts and myoblasts were not affected while normal HUVEC cells promoted CD32A down-regulation. CD32A-CR T cell activity was not inhibited by human IgGs or human serum, but; it was rather enhanced by cetuximab antibody. RNAseq analysis of sensitive vs resistant BC cells identified a fingerprint of 42 genes predicting the sensitivity of BC cells to CD32A-CR T cells and their association with favorable prognostic significance in advanced BC patients. Our data also identify ICAM 1 as a major regulator of CD32A-CR T cell-mediated cytotoxicity. Finally, CD32A-CR T cell administration protected immunodeficient mice from subcutaneous growth of MDA-MB-468 cells in the absence of tumor-specific antibodies. These data indicate that CD32A-CR can be utilized for the identification of (1) cell surface CD32A ligand(s); (2) rational therapeutic strategies to target BC; and (3) novel transcriptomic signatures prognostically relevant for advanced BC patients.


title: NK cells acquire CCR5 and CXCR4 by trogocytosis in people living with HIV-1

abstract:
NK cells play a major role in the antiviral immune response, including against HIV-1. HIV patients have impaired NK cell activity with decrease in CD56dim NK cells and increase in CD56-CD16+ subset and recently it has been proposed that a population of CD56+NKG2C+KIR+CD57+ cells represents antiviral memory NK cells. Antiretroviral therapy (ART) partly restores the functional activity of this lymphocyte lineage. NK cells when interacting with their targets can gain antigens from them by the process of trogocytosis. Here we show that NK cells can obtain CCR5 and CXCR4, but barely CD4, from T cell lines by trogocytosis in vitro. By UMAP (Uniform Manifold Approximation and Projection), we show that aviremic HIV patients have unique NK cell clusters that encompass for cells expressing CCR5, NKG2C and KIRs, but lack CD57 expression. Viremic patients have a larger proportion of CXCR4+ and CCR5+ NK cells than healthy donors (HD) and this is largely increased in CD107+ cells, suggesting a link between degranulation and trogocytosis. In agreement, UMAP identified a specific NK cell cluster in viremic HIV patients, which contains most of the CD107a+, CCR5+ and CXCR4+ cells. However, this cluster lacks NKG2C expression. Therefore, NK cells can gain CCR5 and CXCR4 by trogocytosis, which depends on degranulation.

Author SummaryNK cells play a major role in the antiviral immune response, including against HIV-1. HIV-1 infection largely impacts NK cell lineage. However, identification of the NK cells fighting the virus and eliminating infected cells has remained elusive. By using FACs analysis and analyzing T cell markers, we identify specific NK cell subsets that have gained T cell antigen markers by trogocytosis. We show a strong association of degranulation and trogocytosis ex vivo suggesting that NK cells are eliminating or trying to eliminate HIV-1 infected cells.

Moreover, we show that NK cells can obtain the HIV-1 coreceptors CCR5 and CXCR4, but barely CD4, from T cells. Therefore, by uncovering that NK cells gain CCR5 and CXCR4 by trogocytosis we show that NK cells are actively fighting against HIV-1 infection in viremic and aviremic patients. This should help to identify why certain patients better control the virus, to develop new anti-HIV-1, NK cell-based, immunotherapies and to understand if NK cells can be a new HIV-1 reservoir.


title: ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1

abstract: There is considerable evidence that cross-talk between the Hedgehog pathway and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here, we demonstrate that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. We show that ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for the negative regulator SUFU; furthermore, these phosphorylation events cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, mutant GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.


title: Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

abstract: Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.


title: The transcription factor Zic4 acts as a transdifferentiation switch

abstract:
The molecular mechanisms that maintain cell identities and prevent transdifferentiation remain mysterious. Interestingly, both dedifferentiation and transdifferentiation are transiently reshuffled during regeneration. Therefore, organisms that regenerate readily offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4 silencing is sufficient to induce transdifferentiation of tentacle into foot cells. We identified a Wnt-controlled Gene Regulatory Network that controls a transcriptional switch of cell identity. Furthermore, we show that this switch also controls the re-entry into the cell cycle. Our data indicate that maintenance of cell fate by a Wnt-controlled GRN is a key mechanism during both homeostasis and regeneration.

One-Sentence SummaryA Wnt-controlled GRN controls fate maintenance in Hydra.


title: Ureter single-cell and spatial mapping reveal cell types, architecture, and signaling networks

abstract: Tissue engineering offers a promising treatment strategy for ureteral strictures, but its success requires an in-depth understanding of the architecture, cellular heterogeneity, and signaling pathways underlying tissue regeneration. Here we define and spatially map cell populations within the human ureter using single-cell RNA sequencing, spatial gene expression, and immunofluorescence approaches. We focused on the stromal and urothelial cell populations to enumerate distinct cell types composing the human ureter and inferred potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Furthermore, we analyzed and experimentally validated the importance of Sonic Hedgehog (SHH) signaling pathway in adult stem cell maintenance. The SHH-expressing basal cells supported organoid generation in vitro and accurately predicted the differentiation trajectory from basal stem cells to terminally differentiated umbrella cells. Our results highlight essential processes involved in adult ureter tissue homeostasis and provide a blueprint for guiding ureter tissue engineering.


title: midline represses Dpp signaling and target gene expression in Drosophila ventral leg development.

abstract:
Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. Here we show that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus TBE site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. We further demonstrate that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, we find that mid does not affect all Dpp-target genes as we demonstrate that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.

Summary statementVentral patterning is controlled in part by the T-box Transcription factor midline blocking Dpp signaling and Dpp-activated genes, though midline does not affect the Dpp-repressed gene brk.


title: Lrig1 and Wnt dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis

abstract:
The barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment- producing melanocytes, and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented 'scale' and hypopigmented 'interscale' epidermis. Why and how mature melanocytes accumulate in scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T-cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte- and melanin-independent, and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovered cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

SUMMARY STATEMENTPigmentation and immune surveillance functions in murine tail skin are spatially segregated by Lrig1- and Wnt-Lef1-dependent keratinocyte lineages that control the partitioning of melanocytes and tissue-resident immune cells into distinct epidermal niches.


title: Impact of carbon monoxide on early cardiac development in an avian model

abstract: Carbon monoxide (CO) is a toxic gas that can be lethal in large doses and may also cause physiological damage in lower doses. Epidemiological studies suggest that CO in lower doses over time may impact on embryo development, in particular cardiac development, however other studies have not observed this association. Here, we exposed chick embryos in ovo to CO at three different concentrations (1ppm, 8ppm, 25ppm) plus air control (4 protocols in total) for the first nine days of development, at which point we assessed egg and embryo weight, ankle length, developmental stage, heart weight and ventricular wall thickness. We found that heart weight was reduced for the low and moderate exposures compared to air, and that ventricular wall thickness was increased for the moderate and high exposures compared to air. Ventricular wall thickness was also significantly positively correlated with absolute CO exposures across all protocols. This intervention study thus suggests that CO even at very low levels may have a significant impact on cardiac development.


title: Induced reprogramming of adult murine cardiomyocytes to pluripotency in vivo

abstract: Partial cell reprogramming has been demonstrated in certain mouse tissues by in situ overexpression of Oct3/4, Klf4, Sox2 and cMyc (OKSM) transcription factors, and can trigger rejuvenation and/or augment regeneration of aged or injured tissues. In vivo reprogramming of adult mouse cardiomyocytes has been elusive, but success could overcome the lack of endogenous cardiomyocyte turnover that contributes to the poor resolution of heart disease. Here, we exploited cell type-specific Cre recombination and conditional, doxycycline-inducible, control of gene expression to generate cardiomyocyte-specific, inducible, reprogrammable mice. Eighteen days of doxycycline-induced OKSM expression in this model established a gene expression program characteristic of the pluripotent state and triggered the generation of teratomas of confirmed cardiomyocyte origin. These findings confirm that OKSM reprograms adult mouse cardiomyocytes to pluripotency and will enable studies of the contribution of reprogrammed cardiomyocytes to cardiac regeneration.


title: Habitat deterioration despite protection: long-term declines of littoral area of fishponds in Czech nature reserves

abstract:
Fishponds play a key role in current pondscapes in many developed countries. Their littoral areas, supporting multiple ecosystem functions including the maintenance of aquatic and riparian biodiversity, have been adversely affected by the move shift towards more intensive aquaculture and widespread eutrophication in the middle 20th century. To counteract these changes, many fishponds received some protection, but its long-term efficiency has not been studied. Here we focus on the role of conservation status in protecting the area of littoral areas of fishponds in Czechia between the years 1950 and 2019. We found that the conservation status of these fishponds did not prevent habitat deterioration in most of the fishponds, especially during the second half of the 20th century. Moreover, we detected no significant effects of the reserve establishment year, fishpond area and conservation target on the littoral areas. This suggests that the conservation measures are insufficient across fishpond reserve types. We attribute the negative trends to persisting high fish stocks, especially of common carp, and eutrophication resulting from additional feeding, pond manuring, and ongoing nutrient inputs from the pond catchments. Sediment dredging and high grazing pressure by waterfowl in some reserves can further aggravate the situation. We conclude that effective protection of the littoral areas requires a paradigm shift towards less intensive fish stock management, more frequent summer drainage, and effective reduction of all nutrient inputs to increase the water quality. Such measures can help recover the littoral areas and the associated biota.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/473897v1_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@8e9b31org.highwire.dtl.DTLVardef@6ecaf9org.highwire.dtl.DTLVardef@1cfaefdorg.highwire.dtl.DTLVardef@1e9952f_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LILittoral vegetation in ponds supports high biodiversity but often lacks protection.
C_LIO_LIWe evaluated long-term changes in the littoral areas of fishponds in Czechia.
C_LIO_LIThe areas decreased markedly in both protected and unprotected ponds since 1950.
C_LIO_LIReserve duration, fishpond area and conservation target did not affect the trends.
C_LIO_LIChanges in fishpond management are recommended for littoral area recovery.
C_LI

category: ecology


title: ModelRevelator: Fast phylogenetic model estimation via deep learning

abstract:
Selecting the best model of sequence evolution for a multiple-sequence-alignment (MSA) constitutes the first step of phylogenetic tree reconstruction. Common approaches for inferring nucleotide models typically apply maximum likelihood (ML) methods, with discrimination between models determined by one of several information criteria. This requires tree reconstruction and optimisation which can be computationally expensive. We demonstrate that neural networks can be used to perform model selection, without the need to reconstruct trees, optimise parameters, or calculate likelihoods.

We introduce ModelRevelator, a model selection tool underpinned by two deep neural networks. The first neural network, NNmodelfind, recommends one of six commonly used models of sequence evolution, ranging in complexity from JC to GTR. The second, NNalphafind, recommends whether or not a{Gamma} --distributed rate heterogeneous model should be incorporated, and if so, provides an estimate of the shape parameter, . Users can simply input an MSA into ModelRevelator, and swiftly receive output recommending the evolutionary model, inclusive of the presence or absence of rate heterogeneity, and an estimate of .

We show that ModelRevelator performs comparably with likelihood-based methods over a wide range of parameter settings, with significant potential savings in computational effort. Further, we show that this performance is not restricted to the alignments on which the networks were trained, but is maintained even on unseen empirical data. ModelRevelator will be made freely available in the forthcoming version of IQ-Tree (http://www.iqtree.org), and we expect it will provide a valuable alternative for phylogeneticists, especially where traditional methods of model selection are computationally prohibitive.

category: evolutionary biology


title: Overcoming a 'forbidden phenotype': The parrot's head supports, propels, and powers tripedal locomotion

abstract: No vertebrate, living or extinct, is known to have possessed an odd number of limbs. Despite this "forbidden phenotype", gaits that utilize odd numbers of limbs (e.g., tripedalism or pentapedalism) have evolved in both avian and mammalian lineages. Tripedal locomotion is commonly employed by parrots during climbing, who utilize their beaks as an additional support. However, it is unclear whether the beak functions simply as a stabilizing hook, or as a propulsive limb. Here, we present data on kinetics of tripedal climbing in six rosy-faced lovebirds (Agapornis rosiecollis). Our findings demonstrate that parrots utilize cyclical tripedal gaits when climbing and the beak and hindlimbs generate comparable propulsive and tangential substrate reaction forces and power. Propulsive and tangential forces generated by the beak are of equal or greater magnitudes to those forces generated by the forelimbs of humans and non-human primates during vertical climbing. We conclude that the feeding apparatus and neck musculature of parrots has been co-opted to function biomechanically as a third limb during vertical climbing. We hypothesize that this exaptation required substantive alterations to the neuromuscular system including enhanced force-generating capabilities of the neck musculature and modifications to limb central pattern generators.


title: HuluFISH non-denaturing in situ detection of genomic DNA opened by CRISPR-Cas9 Nickase and Exonuclease

abstract: DNA fluorescence in situ hybridization (FISH) has been widely used in diagnosis and genetic research. Traditional Bacterial artificial chromosome (BAC) or oligonucleotide based probe to detect DNA in situ is only effective when the target is relatively large, usually over 150Kb DNA fragments. And it involves heat denaturation steps to open the DNA for in situ hybridization. The heat process can affect the fine structure of nuclei. Here we reported a novel method based on Cas9 nickase and exonuclease digestion of double strand DNA and permanently mark the DNA in single strand state for FISH. With this novel design, we detected non-repetitive genomic loci as small as 2Kb.

category: genomics


title: Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish

abstract: Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.


title: Transcriptome-wide analysis of microRNA-mRNA correlations in unperturbed tissue transcriptomes identifies microRNA targeting determinants.

abstract: MicroRNAs are small RNAs that regulate gene expression through complementary base pairing with their target mRNAs. Given the small size of the pairing region and the large number of mRNAs that each microRNA can control, the identification of biologically relevant targets is difficult. Since current knowledge of target recognition and repression has mainly relied on in vitro studies, we sought to determine if the interrogation of gene expression data of unperturbed tissues could yield new insight into these processes. The transcriptome-wide repression at the microRNA-mRNA canonical interaction sites (seed and 3'-supplementary region, identified by sole base complementarity) was calculated as a normalized Spearman correlation (Z-score) between the abundance of the transcripts in the PRAD-TCGA tissues (RNA-seq and small RNA-seq data of 546 samples). Using the repression values obtained we confirmed established properties or microRNA targeting efficacy, such as the preference for gene regions (3UTR>CDS>5UTR), the proportionality between repression and seed length (6mer<7mer<8mer) and the contribution to the repression exerted by the supplementary pairing at 13-16nt of the microRNA. Our results suggest that the 7mer-m8 seed could be more repressive than the 7mer-A1, while they have similar efficacy when they interact using the 3-supplementary pairing. Strikingly, the 6mer+suppl sites yielded normalized Z-score of repression similar to the sole 7mer-m8 or 7mer-A1 seeds, which raise awareness of its potential biological relevance. We then used the approach to further characterize the 3-supplementary pairing, using 39 microRNAs that hold repressive 3-supplementary interactions. The analysis of the bridge between seed and 3-supplementary pairing site confirmed the optimum +1 offset previously evidenced, but higher offsets appear to hold similar repressive strength. In addition, they show a low GC content at position 13-16, and base preferences that allow the selection of a candidate sequence motif. Overall, our study demonstrates that transcriptome-wide analysis of microRNA-mRNA correlations in large, matched RNA-seq and small-RNA-seq data has the power to uncover hints of microRNA targeting determinants operating in the in vivo unperturbed set. Finally, we made available a bioinformatic tool to analyze microRNA-target mRNA interactions using our approach.


title: Characterization of antibiotic resistance in clinical isolates of Klebsiella pneumoniae in Denmark

abstract: Klebsiella pneumoniae (KP) is a major global health problem as it leads to hospital outbreaks all over the world and is becoming more difficult to treat due to its increasing antimicrobial resistance (AMR). Optimization and development of new treatments of KP requires understanding of its population structure and AMR properties. Therefore, in this study, we collected and sequenced 491 KP strains from four major Danish microbiology departments covering 51% of the Danish population. The isolates were whole genome sequenced (WGS), phenotypically characterized and compared with 2,124 KP strains from 13 different countries (PATRIC strains). We found that while genomic content varies significantly across the Danish strains, they also differ significantly from strains from other countries, due to the lack of certain AMR sequence types (e.g. ST258 and ST307) in Denmark. Genomic and experimental analysis suggest that Danish strains contain fewer virulence mechanisms and are more susceptible to antimicrobials compared to strains from other countries, likely due to the relatively low antibiotic usage in Denmark where 70% of hospital antibiotic usage is penicillins. We also identified potential novel AMR determinants to tigecycline through statistical analysis of genomic and phenotypic data. To conclude, we obtained a more comprehensive understanding of the KP strains in Denmark and provided valuable insights for future experiments and strategies to combat AMR in KP.


title: Putting perception into action: Inverse optimal control for continuous psychophysics

abstract: Psychophysical methods are a cornerstone of psychology, cognitive science, and neuroscience where they have been used to quantify behavior and its neural correlates for a vast range of mental phenomena. Their power derives from the combination of controlled experiments and rigorous analysis through signal detection theory. Unfortunately, they require many tedious trials and preferably highly trained participants. A recently developed approach, continuous psychophysics, promises to transform the field by abandoning the rigid trial structure involving binary responses and replacing it with continuous behavioral adjustments to dynamic stimuli. However, what has precluded wide adoption of this approach is that current analysis methods recover perceptual thresholds, which are one order of magnitude larger compared to equivalent traditional psychophysical experiments. Here we introduce a computational analysis framework for continuous psychophysics based on Bayesian inverse optimal control. We show via simulations and on previously published data that this not only recovers the perceptual thresholds but additionally estimates subjects action variability, internal behavioral costs, and subjective beliefs about the experimental stimulus dynamics. Taken together, we provide further evidence for the importance of including acting uncertainties, subjective beliefs, and, crucially, the intrinsic costs of behavior, even in experiments seemingly only investigating perception.

category: animal behavior and cognition


title: Reconfigurable microfluidic circuits for isolating and retrieving cells of interest

abstract: Microfluidic devices are widely used in many fields of biology, but a key limitation is that cells are typically surrounded by solid walls, making it hard to access those that exhibit a specific phenotype for further study. Here, we provide a general and flexible solution to this problem that exploits the remarkable properties of microfluidic circuits with fluid walls - transparent interfaces between culture media and an immiscible fluorocarbon that are easily pierced with pipets. We provide two proofs-of-concept in which specific cell sub-populations are isolated and recovered: i) murine macrophages chemotaxing towards complement component 5a, and ii) bacteria (Pseudomonas aeruginosa) in developing biofilms that migrate towards antibiotics. We build circuits in minutes on standard Petri dishes, add cells, pump in laminar streams so molecular diffusion creates attractant gradients, acquire time-lapse images, and isolate desired sub-populations in real-time by building fluid walls around migrating cells with an accuracy of tens of micrometres using 3D-printed adaptors that convert conventional microscopes into wall-building machines. Our method allows live cells of interest to be easily extracted from microfluidic devices for downstream analyses.

category: bioengineering


title: An analytical solution simulating growth of Lewy bodies

abstract: This paper reports a minimal model simulating the growth of a Lewy body (LB). To the best of our knowledge, this is the first model simulating Lewy body growth. The LB is assumed to consist of a central spherical core, which is composed of membrane fragments and various dysfunctional intracellular organelles, and a halo, which is composed of alpha-synuclein fibrils. Membrane fragments and alpha-synuclein monomers are assumed to be produced in the soma at constant rates. The growth of the core and the halo are simulated by the Finke-Watzky model. Analytical (closed-form) solutions describing the growth of the core and the halo are obtained. A sensitivity analysis in terms of model parameters is performed.


title: Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy

abstract: Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously due to spectral overlap. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, we describe a new technique called Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS), that integrates SRS microscopy with expansion microscopy (ExM). ExM is a powerful strategy providing significant improvement in imaging resolution by physical magnification of hydrogel-embedded preserved biological specimens. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. We used MAGNIFIERS to visualize nanoscale features in a label-free manner with C-H vibration of proteins, lipids and DNA in a broad range of biological specimens, from mouse brain, liver and kidney to human lung organoid. In addition, we applied MAGNIFIERS to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, we used MAGNIFIERS to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.