title: SSAM-lite: a light-weight web app for rapid analysis of spatially resolved transcriptomics data

abstract:
1The combination of a cells transcriptional profile and location defines its function in a spatial context. Spatially resolved transcriptomics (SRT) has emerged as the assay of choice for characterizing cells in situ. SRT methods can resolve gene expression up to single-molecule resolution. A particular computational problem with single-molecule SRT methods is the correct aggregation of mRNA molecules into cells. Traditionally, aggregating mRNA molecules into cell-based features begins with the identification of cells via segmentation of the nucleus or the cell membrane. However, recently a number of cell-segmentation-free approaches have emerged. While these methods have been demonstrated to be more performant than segmentation-based approaches, they are still not easily accessible since they require specialized knowledge of programming languages and access to large computational resources. Here we present SSAM-lite, a tool that provides an easy-to-use graphical interface to perform rapid and segmentation-free cell-typing of SRT data in a web browser. SSAM-lite runs locally and does not require computational experts or specialized hardware. Analysis of a tissue slice of the mouse somatosensory cortex took less than a minute on a laptop with modest hardware. Parameters can interactively be optimized on small portions of the data before the entire tissue image is analyzed. A server version of SSAM-lite can be run completely offline using local infrastructure. Overall, SSAM-lite is portable, lightweight, and easy to use, thus enabling a broad audience to investigate and analyze single-molecule SRT data.

Availability and ImplementationSSAM-lite is an open-source browser-based web application with source code freely available on Github via https://github.com/HiDiHlabs/ssam-lite. Stable releases can be accessed via https://ssam-lite.bihealth.org and https://ssam-lite.netlify.app, and developmental releases can be accessed via https://dev--ssam-lite.netlify.app. The source code for a locally deployable server version, SSAM-lite-server, is available on GitHub via https://github.com/HiDiHlabs/ssam-lite-server. Both versions require a modern browser with JavaScript and WebGL support. Detailed user guides and documentation can be found at https://ssam-lite.readthedocs.io.


title: A Pre-Processing Pipeline to Quantify, Visualize andReduce Technical Variation in Protein Microarray

abstract:
Technical variation, or variation from non-biological sources, is present in most laboratory assays. Correcting for this variation enables analysts to extract a biological signal that informs questions of interest. However, each assay has different sources and levels of technical variation and the choice of correction methods can impact downstream analyses. Compared to similar assays such as DNA microarrays, relatively few methods have been developed and evaluated for protein microarrays, a versatile tool for measuring levels of various proteins in serum samples. Here, we propose a pre-processing pipeline to correct for some common sources of technical variation in protein microarrays. The pipeline builds upon an existing normalization method by using controls to reduce technical variation. We evaluate our method using data from two protein microarray studies, and by simulation. We demonstrate that pre-processing choices impact the fluorescent-intensity based ranks of proteins, which in turn, impact downstream analysis.

1 Impact StatementProtein microarrays are in wide use in cancer research, infectious disease diagnostics and biomarker identification. To inform research and practice in these and other fields, technical variation must be corrected using normalization and pre-processing. Current protein microarray studies use a variety of normalization methods, many of which were developed for DNA microarrays, and therefore are based on assumptions and data that are not ideal for protein microarrays. To address this issue, we develop, evaluate, and implement a pre-processing pipeline that corrects for technical variation in protein microarrays. We show that pre-processing and normalization directly impact the validity of downstream analysis, and protein-specific approaches are essential.

category: bioinformatics


title: Computational design of highly signaling active membrane receptors through de novo solvent-mediated allosteric networks

abstract: Protein catalysis and allostery require the atomic-level orchestration and motion of residues, ligand, solvent and protein effector molecules, but the ability to design protein activity through precise protein-solvent cooperative interactions has not been demonstrated. Here, we report the design of a dozen novel membrane receptors catalyzing G-protein nucleotide exchange through diverse de novo engineered allosteric pathways mediated by cooperative networks of intra-protein, protein-ligand and solvent molecule interactions. Consistent with the predictions, designed protein activities strongly correlated with the level of solvent-mediated interaction network plasticity at flexible transmembrane helical interfaces. Several designs displayed considerably enhanced thermostability and activity compared to related natural receptors. The most stable and active variant crystallized in an unforeseen signaling active conformation, in excellent agreement with the design models. The allosteric network topologies of the best designs bear limited similarity to those of natural receptors and reveal a space of allosteric interactions larger than previously inferred from natural proteins. The approach should prove useful for engineering proteins with novel complex protein catalytic and signaling activities.

category: biophysics


title: Sex Differences in Brain Tumor Glutamine Metabolism Reveal Sex-Specific Vulnerabilities to Treatment

abstract: Sex differences in normal metabolism are well described, but whether they persist in cancerous tissue is unknown. We assessed metabolite abundance in glioblastoma surgical specimens and found that male glioblastomas are enriched for amino acids, including glutamine. Using PET imaging, we found that gliomas in male patients exhibit significantly higher glutamine uptake. These sex differences were well-modeled in murine transformed astrocytes, in which male cells imported and metabolized more glutamine and were more sensitive to glutaminase 1 (GLS1) inhibition. The sensitivity to GLS1 inhibition in males was driven by their dependence on glutamine-derived glutamate for -ketoglutarate synthesis and TCA cycle replenishment. Females were resistant to GLS1 inhibition through greater pyruvate carboxylase-mediated TCA cycle replenishment. Thus, clinically important sex differences exist in targetable elements of metabolism. Recognition of sex-biased metabolism is an opportunity to improve treatments for all patients through further laboratory and clinical research.

category: cancer biology


title: AtCGL160 recruits chloroplast coupling factor 1

abstract: ATP synthases couple the generation of chemical energy to a transmembrane electro-chemical potential. Like ATP synthases in bacteria and mitochondria, chloroplast ATP synthases consist of a membrane-spanning (CFO) and a soluble coupling factor (CF1). Accessory factors facilitate subunit production and orchestrate the assembly of the functional CF1-CFO complex. It was previously shown that the accessory factor CGL160 promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. In this study, we show that the N-terminal portion of CGL160 (AtCGL160N), which is specific to the green lineage, is required for late steps in CF1-CFO assembly in Arabidopsis thaliana. In plants that lacked this stroma-exposed domain, photosynthesis was impaired, and amounts of CF1-CFO were reduced to about 65% of the wild-type level. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of CF1 sub-complexes in the stroma relative to the wild type and the CF1 assembly mutant atcgld11-1. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the thylakoid-proximal domain of CF1-{beta} that includes the conserved DELSEED motif. We therefore propose that AtCGL160 has acquired an additional function in the recruitment of soluble CF1 to a membrane-integral CFO sub-complex, which is critical for the modulation of CF1-CFO activity and photosynthesis in chloroplasts.

category: plant biology


title: Why can we detect lianas from space?

abstract: Lianas are found in virtually all tropical forests and have strong impacts on the forest carbon cycle by slowing tree growth, increasing tree mortality and arresting forest succession. In a few local studies, ecologists have successfully differentiated lianas from trees using various remote sensing platforms including satellite images. This demonstrates a potential to use remote sensing to investigate liana dynamics at spatio-temporal scales beyond what is currently possible with ground-based inventory censuses. However, why do liana-infested tree crowns and forest stands display distinct spectral signals? And is the spectral signal of lianas only locally unique or consistent across continental and global scales? Unfortunately, we are not yet able to answer these questions, and without such an understanding the limitations and caveats of large-scale application of automated classifiers cannot be understood. Here, we tackle the questions of why we can detect lianas from airborne and spaceborne remote sensing platforms. We identify whether a distinct spectral distribution exists for lianas, when compared to their tree hosts, at the leaf, canopy and stand scales in the solar spectrum (400 to 2500 nm). To do so, we compiled databases of (i) leaf reflectance spectra for over 4771 individual leaves of 539 species, (ii) fine-scale ([~]1m2) surface reflectance from 999 tree canopies characterized by different levels of liana infestation in Panama and Malaysia, and (iii) coarse-scale (>100 m2) surface reflectance from hundreds of hectares of heavily infested liana forest stands in French Guiana and Bolivia. Using these data, we find consistent spectral signal of liana-infested canopies across sites with a mean inter-site correlation of 89% (range 74-94%). However, as we find no consistent difference between liana and tree leaves, a distinct liana spectral signal appears to only manifests at the canopy and stand scales (>1m2). To better understand this signal, we implement mechanistic radiative transfer models capable of modeling the vertically stratificatied non-linear mixing of spectral signals intrinsic to lianas infestation of forest canopies. Next, we inversely fit the models to observed spectral signals of lianas at all scales to identify key biochemical or biophysical processes. We then corroborate our model results with field data on liana leaf chemistry and canopy structural properties. Our results suggest that a liana-specific spectral distribution arises due to the combination of cheaply constructed leaves and efficient light interception. A model experiment revealed that the spectral distribution was most sensitive to lower leaf and water mass per unit area, affecting the absorption of NIR and SWIR radiation, and a more planophile (flatter) leaf angle distribution. Finally, we evaluate the theoretical discernibility of lianas from trees and how this varies with remote sensing platforms and resolution. We end by discussing the potential, limitations and risks of applying automated classifiers to detect lianas from remotely sensed data at large scales.


title: A deep learning algorithm for potato tuber hollowheart classification

abstract: A novel deep learning algorithm is proposed for hollow heart detection which is an internal tuber defect. Hollow heart is one of many internal defects that decrease the market value of potatoes in the fresh market and food processing sectors. Susceptibility to internal defects like the hollow heart is influenced by genetic and environmental factors so elimination of defect-prone material in potato breeding programs is important. Current methods of evaluation utilize human scoring which is limiting (only collects binary data) relative to the data collection capacity afforded by computer vision or are based upon X-ray transmission techniques that are both expensive and can be hazardous. Automation of defect classification (e.g. hollow heart) from data sets collected using inexpensive, consumer-grade hardware has the potential to increase throughput and reduce bias in public breeding programs. The proposed algorithm consists of ResNet50 as the backbone of the model followed by a shallow fully connected network (FCN). A simple augmentation technique is performed to increase the number of images in the data set. The performance of the proposed algorithm is validated by investigating metrics such as precision and the area under the curve (AUC).


title: arakno - An R package for effective spider nomenclature, distribution and trait data retrieval from online resources

abstract: Online open databases are increasing in number, usefulness, and ease of use. There are currently two main global databases exclusive for spiders, the World Spider Catalogue (WSC) and the World Spider Trait (WST) database. Both are regularly used by thousands of researchers. Computational tools that allow effective processing of large data are now part of the workflow of any researcher and R is becoming a de facto standard for data manipulation, analysis, and presentation. Here we present an R package, arakno, that allows interface with the two databases. Implemented tools include checking species names against nomenclature of the WSC, obtaining and mapping data on distribution of species from both WST and the Global Biodiversity Information Facility (GBIF), and downloading trait data from the WST. A set of tools are also provided to prepare data for further statistical analysis.

category: zoology


title: The organization of individually mapped structural and functional semantic networks in aging adults

abstract: Language function in the brain, once thought to be highly localized, is now appreciated as relying on a connected but distributed network. The semantic system is of particular interest in the language domain because of its hypothesized integration of information across multiple cortical regions. Previous work in healthy individuals has focused on group-level functional connectivity (FC) analyses of the semantic system, which may obscure interindividual differences driving variance in performance. These studies also overlook the contributions of white matter networks to semantic function. Here, we identified semantic network nodes with a semantic decision fMRI task in 53 typically-aging adults, characterized network organization using structural connectivity (SC), and quantified the segregation and integration of the network using FC. Hub regions were identified in left inferior frontal gyrus. The individualized semantic network was composed of three interacting modules: 1) default-mode module characterized by bilateral medial prefrontal and posterior cingulate regions and also including right-hemisphere homotopes of language regions; 2) left frontal module extending dorsally from inferior frontal gyrus to pre-motor area; and 3) left temporoparietal module extending from temporal pole to inferior parietal lobule. FC within Module3 and integration of the entire network related to a semantic verbal fluency task, but not a matched phonological task. These results support and extend the tri-network semantic model (Xu et al., 2017) and the controlled semantic cognition model (Chiou et al., 2018) of semantic function.

category: neuroscience


title: Odor motion sensing enables complex plume navigation

abstract: Studies dating back a century (Flugge, 1934) have stressed the critical role of the wind as the primary directional cue in odor plume navigation. Here, we show that Drosophila shape their navigational decisions using a second directional cue - the direction of motion of odors - which they detect from the temporal correlations of the odor signal between their two antennae. Using a high-resolution virtual reality paradigm to deliver spatiotemporally complex fictive odors to freely-walking flies, we demonstrate that such odor direction sensing is computationally equivalent to motion detection algorithms underlying motion detection in vision. Simulations and theoretical analysis of turbulent plumes reveal that odor motion contains valuable directional information absent from the airflow; indeed, this information is used by both Drosophila and virtual agents to navigate naturalistic odor environments. The generality of our findings suggests that odor direction sensing is likely used throughout the animal kingdom, and could significantly improve olfactory robot navigation in harsh chemical environments.


title: Towards real-world neuroscience using mobile EEG and augmented reality

abstract: Our visual environment impacts multiple aspects of cognition including perception, attention and memory, yet most studies traditionally remove or control the external environment. As a result, we have a limited understanding of neurocognitive processes beyond the controlled lab environment. Here, we aim to study neural processes in real-world environments, while also maintaining a degree of control over perception. To achieve this, we combined mobile EEG (mEEG) and augmented reality (AR), which allows us to place virtual objects into the real world. We validated this AR and mEEG approach using a well-characterised cognitive response - the face inversion effect. Participants viewed upright and inverted faces in three EEG tasks (1) a lab-based computer task, (2) walking through an indoor environment while seeing face photographs, and (3) walking through an indoor environment while seeing virtual faces. We find greater low frequency EEG activity for inverted compared to upright faces in all experimental tasks, demonstrating that cognitively relevant signals can be extracted from mEEG and AR paradigms. This was established in both an epoch-based analysis aligned to face events, and a GLM-based approach that incorporates continuous EEG signals and face perception states. Together, this research helps pave the way to exploring neurocognitive processes in real-world environments while maintaining experimental control using AR.


title: The proSAAS chaperone provides neuroprotection and attenuates transsynaptic α-synuclein spread in rodent models of Parkinson's disease

abstract:
Parkinsons disease is a devastating motor disorder involving the aberrant aggregation of the synaptic protein synuclein (aSyn) and degeneration of the nigrostriatal dopaminergic tract. We previously showed that proSAAS, a small secreted chaperone protein widely expressed in neurons within the brain, is able to block aSyn-induced dopaminergic cytotoxicity in primary nigral neuron cultures. We show here that coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This positive functional outcome was accompanied by significant amelioration of the human aSyn-induced loss of both nigral tyrosine hydroxylase-positive cells and striatal tyrosine hydroxylase-positive terminals, demonstrating clear proSAAS-mediated protection of the nigro-striatal tract. ProSAAS overexpression also reduced the content of human aSyn protein in both the nigra and striatum and reduced the loss of tyrosine hydroxylase protein in both regions. Since proSAAS is a secreted protein, we tested the possibility that proSAAS is able to block the transsynaptic spread of aSyn from the periphery to the central nervous system, increasingly recognized as a potentially significant pathological mechanism. The number of human aSyn-positive neurites in the pons and caudal midbrain of mice following administration of human aSyn-encoding AAV into the vagus nerve was considerably reduced in mice coinjected with proSAAS-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. We suggest that proSAAS may represent a promising target for therapeutic development in Parkinsons disease.

SignificanceThis paper describes two independent avenues of research that both provide support for the in vivo neuroprotective function of this small chaperone protein. In the first approach, we show that proSAAS overexpression provides remarkably effective protection against dopaminergic neurotoxicity in a rat model of Parkinsons disease. This conclusion is supported both by three independent assays of motor function as well as by quantitative analysis of surviving dopaminergic neurons in brain areas involved in the control of motor function. In the second line of research, we show that in mice, the spread of human synuclein across synapses can be blunted by proSAAS overexpression.


title: A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

abstract: miRNAs are regulatory transcripts established as repressors of mRNA stability and translation. Here we demonstrate that a key onco-miRNA, miR-10b, binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of direct binding between miR-10b and U6, in situ visualizations of miR-10b and U6 co-localization in glioma cells and patient-derived tumor tissues, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in splicing alterations, illustrated by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

category: molecular biology


title: KRAB Zinc Finger protein Znf684 interacts with Nxf1 to regulate mRNA export

abstract: Cys2His2 (C2H2) type zinc finger (ZnF) proteins constitute a large class of proteins that are generally considered to be DNA-binding transcription factors. Using affinity purification followed by mass spectrometry, as well as reciprocal co-immunoprecipitation experiments, we determined that the C2H2-ZnF protein Znf684 interacts physically with several proteins involved in mRNA export, including Nxf1 and Alyref. We utilized individual nucleotide resolution cross-linking immunoprecipitation followed by high throughput sequencing (iCLIP-seq) experiments to show that Znf684 binds directly to specific mRNAs in vivo and has an RNA-binding profile similar to those of Nxf1 and Alyref, suggesting a role in mRNA export regulation. Immunofluorescence microscopy (IF) experiments revealed that Znf684 localizes to both the nucleus and cytoplasm. Using cellular fractionation experiments, we demonstrate that overexpression of Znf684 negatively impacts the export of SMAD3 and other target mRNAs. Taken together, our results suggest that Znf684 regulates the export of a subset of transcripts through physical interactions with Nxf1 and specific target mRNAs.


title: FGF signalling is involved in cumulus migration in the common house spider Parasteatoda tepidariorum

abstract:
Cell migration is a fundamental component during the development of most multicellular organisms. In spiders, the collective migration of a signalling centre, known as the cumulus, is required to set the dorsoventral body axis of the embryo.

Here, we show that FGF signalling plays an important role during cumulus migration in the spider Parasteatoda tepidariorum. Spider embryos with reduced FGF signalling lack cumulus migration and display dorsoventral patterning defects. Our study reveals that cumulus expression of several FGF signalling components is regulated by the transcription factor Ets4. In conjunction with a previous study, we show that the expression of fgf8 in the germ-disc is regulated via the Hedgehog signalling pathway. We also demonstrate that FGF signalling influences the BMP signalling pathway activity in the region around cumulus cells.

Finally, we show that FGF signalling might also influence cumulus migration in basally branching spiders and we propose a hypothetical model in which fgf8 acts a chemo-attractant to guide cumulus cells towards the future dorsal pole of the spider embryo.

category: developmental biology


title: A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant-negative impact on CSF1R signaling.

abstract:
Amino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout; with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+ mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein which expands tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborization were reduced in the Csf1rE631K/+ mice as in ALSP patients. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that disease-associated CSF1R mutations encode dominant negative repressors of CSF1R signaling. We speculate that leukoencephalopathy associated with human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles.

Summary StatementThis study describes the effect of a human disease-associated mutation in the mouse CSF1R gene on postnatal development and growth factor responsiveness of cells of the macrophage lineage.


title: Fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm

abstract:
The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, Congenital Diaphragmatic Hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors or potentially the phrenic nerve have not been explicitly tested. Using conditional mutagenesis and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue, are the source of HGF critical for diaphragm muscularization and phrenic nerve primary branching. HGF not only is required for recruitment of muscle progenitors to the diaphragm, but is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Thus, the connective tissue fibroblasts and HGF coordinately regulate diaphragm muscularization and innervation. Defects in PPF-derived HGF result in muscleless regions that are susceptible to CDH.

Summary StatementFibroblast-derived HGF signals to Met+ muscle progenitors and nerve to control the expansion of diaphragm muscle and primary branching of phrenic nerve axons - structures critical for breathing in mammals.


title: Plant-insect chemical communication in ecological communities: an information theory perspective

abstract: Cross-species communication, where signals are sent by one species and perceived by others, is one of the most intriguing types of communication that functionally links different species to form complex ecological networks. Yet, global changes and human activities can affect communication by increasing the fluctuations of species composition and phenology, altering signal profiles and intensity, and introducing noises. So far, most studies on cross-species communication have focused on a few specific species isolated from ecological communities. Scaling up investigations of cross-species communication to the community level is currently hampered by a lack of conceptual and practical methodologies. Here, we propose an interdisciplinary framework based on information theory to investigate mechanisms shaping cross-species communication at the community level. We use plants and insects, the cornerstones of most ecosystems, as a showcase; and focus on chemical communication as the key communication channel. We first introduce some basic concepts of information theory, then we illustrate information patterns in plant-insect chemical communication, followed by a further exploration of how to integrate information theory into ecological and evolutionary processes to form testable mechanistic hypotheses. We conclude by highlighting the importance of community-level information as a vehicle to better understand the maintenance of ecological systems, especially when facing rapid global changes.

category: ecology


title: Plasticity in female timing may explain current shifts in breeding phenology of a North American songbird

abstract:
O_LIClimate change has driven changes in breeding phenology. Identifying the magnitude of phenological shifts and whether selection in response to climate change drives these shifts is key for determining species reproductive success and persistence in a changing world.
C_LIO_LIWe investigated reproductive timing in a primarily sedentary population of the dark-eyed junco (Junco hyemalis) over 32 years. We predicted that juncos would breed earlier in warmer springs in response to selection favouring earlier breeding.
C_LIO_LITo test this prediction, we compared the annual median date for reproductive onset (i.e., egg one date) to monthly spring temperatures and examined evidence for selection favouring earlier breeding and for plasticity in timing.
C_LIO_LIEgg one dates occurred earlier over time, with the timing of breeding advancing up to 24 days over the 32-year period. Breeding timing also strongly covaried with maximum April temperature. We found significant overall selection favouring earlier breeding (i.e., higher relative fitness with earlier egg one dates) that became stronger over time, but strength of selection was not predicted by temperature. Lastly, individual females exhibited plastic responses to temperature across years.
C_LIO_LIOur findings provide further evidence that phenotypic plasticity plays a crucial role in driving phenological shifts in response to climate change. For multi-brooded bird populations, a warming climate might extend the breeding season and provide more opportunities to re-nest rather than drive earlier breeding in response to potential phenological mismatches. However, as plasticity will likely be insufficient for long-term survival in the face of climate change, further research in understanding the mechanisms of female reproductive timing will be essential for forecasting the effects of climate change on population persistence.
C_LI


title: Deficiency of ZC3HC1 increases vascular smooth muscle cell migration, proliferation and neointima formation following injury

abstract:
The ZC3HC1 gene is associated with various cardiovascular traits in that its common missense variant, rs11556924-T (p.Arg363His), lowers risk of coronary artery disease (CAD) and blood pressure, but increases carotid intima-media thickness (IMT). This study was designed to determine the mechanisms by which ZC3HC1 modulates IMT using in vitro and in vivo models.

We assessed the effect of the rs11556924-T allele on ZC3HC1 expression in vascular smooth muscle cells (SMCs) from 151 multi-ethnic heart transplant donors and found that rs11556924-T was significantly associated with lower ZC3HC1 expression and faster SMC migration. These results were supported by in vitro silencing experiments. At the protein level, ZC3HC1 deficiency resulted in the accumulation of cyclin B1, a key cell cycle protein. Further, transcriptome analysis revealed changes in the regulation of canonical SMC marker genes, including ACTA2, CNN1, LMOD1, and TAGLN. Pathway analysis of differentially expressed genes in SMCs secondary to ZC3HC1 knockdown showed decreased expression of genes in the cell division and cytoskeleton organization pathways.

In line, primary SMCs isolated from the aortas of Zc3hc1-/- mice migrated faster and proliferated more compared to SMCs isolated from wild-type littermates, with the former also showing accumulation of cyclin B1. Neointima formation was also enhanced in Zc3hc1-/- mice in response to arterial injury mimicking restenosis.

Taken together, these findings demonstrate that genetic modulation or deficiency of ZC3HC1 leads to the accumulation of cyclin B1 in SMCs and increased migration, proliferation, and injury-induced neointima formation. We further discuss and propose that a genetic variant regulating SMC proliferation may enhance IMT and early atherosclerosis progression but may be beneficial for plaque stability in advanced lesions.

category: genetics


title: CoCoRV: a rare variant analysis framework using publicly available genotype summary counts to prioritize germline disease-predisposition genes

abstract: Sequencing cases without matched healthy controls hinders prioritization of germline disease-predisposition genes. To circumvent this problem, genotype summary counts from public data sets can serve as controls. However, systematic inflation and false positives can arise if confounding factors are not addressed. We propose a new framework, consistent summary counts based rare variant burden test (CoCoRV), to address these challenges. CoCoRV has consistent variant quality control and filtering, ethnicity-stratified rare variant association test, accurate estimation of inflation factors, powerful FDR control, and can detect rare variants in high linkage disequilibrium. When we applied CoCoRV to pediatric cancer cohorts, the top genes identified were cancer-predisposition genes. We also applied CoCoRV to identify disease-predisposition genes in adult brain tumors and amyotrophic lateral sclerosis. Given that potential confounding factors were well controlled after applying the framework, CoCoRV provides a cost-effective solution to prioritizing disease-risk genes enriched with rare pathogenic variants.


title: Large-scale multi-omics analysis suggests specific roles for intragenic cohesin in transcriptional regulation

abstract: Cohesin, an essential protein complex for chromosome segregation, regulates transcription through a variety of mechanisms. It is not a trivial task to genome-widely assign the diverse cohesin functions. Moreover, the context-specific roles of cohesin-mediated interactions, especially on intragenic regions, have not been thoroughly investigated. Here we performed a comprehensive characterization of cohesin binding sites in several human cell types. We integrated epigenomic, transcriptomic and chromatin interaction data with and without transcriptional stimulation, to explore context-specific functions of intragenic cohesin related to gene activation. We identified a new subset of cohesin binding sites, decreased intragenic cohesin sites (DICs), which have a different function from previously known ones. The intron-enriched DICs were negatively correlated with transcriptional regulation: a subgroup of DICs were related to enhancer markers and paused RNA polymerase II, whereas others contributed to chromatin architecture. We implemented machine learning and successfully isolated DICs with distinct genomic features. We observed DICs in various cell types, including cells from cohesinopathy patients. These results suggest a previously unidentified function of cohesin at intragenic regions for transcription regulation.

category: genomics


title: Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling

abstract: Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probe pools targeting the protein-coding genes in the human and mouse transcriptomes, hereafter referred to as the human or mouse Whole Transcriptome Atlas (WTA). We validated the human and mouse WTA using cell lines to demonstrate concordance with orthogonal gene expression profiling methods in profiled region sizes ranging from ~10-500 cells. By benchmarking against bulk RNAseq and fluorescence in situ hybridization, we demonstrate robust transcript detection possible down to ~100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. We show that spatial profiling with WTA can detect expected spatial gene expression differences between tumor and tumor microenvironment, identify spatial disease-specific heterogeneity in gene expression in histological structures of the human kidney, and comprehensively map transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.


title: Single-cell and nucleus RNA-seq in a mouse model of AD reveal activation of distinct glial subpopulations in the presence of plaques and tangles

abstract: Multiple mouse models have been generated that strive to recapitulate human Alzheimers disease (AD) pathological features to investigate disease mechanisms and potential treatments. The 3xTg-AD mouse presents the two major hallmarks of AD, which are plaques and tangles that increase during aging. While behavioral changes and the accumulation of plaques and tangles have been well described in the 3xTg-AD mice, the subpopulations of neurons and glial cells present throughout disease progression have not been characterized. Here, we used single-cell RNA-seq to investigate changes in subpopulations of microglia, and single-nucleus RNA-seq to explore subpopulations of neurons, astrocytes, and oligodendrocytes in the hippocampus and cortex of aging 3xTg-AD as well as 5xFAD mice for comparison. We recovered a common path of age-associated astrocyte activation between the 3xTg-AD and the 5xFAD models and found that 3xTg-AD-derived astrocytes seem to be less activated. We identified multiple subtypes of microglia, including a subpopulation with a distinct transcription factor expression profile that showed an early increase in Csf1 expression before the switch to disease associated microglia (DAM). We used bulk RNA-seq in the hippocampus of 3xTg-AD mice across their lifespan to identify distinct modules of genes whose expression increases with aging and worsening pathology. Finally, scATAC-seq revealed multiple subpopulations of cells with accessible chromatin in regions around genes associated with glial activation. Overall, differences between the main glial groups point to a slower activation process in the 3xTg-AD model when compared to the 5xFAD. Our study contributes to the identification of progressive transcriptional changes of glial cells in a mouse model that has plaques and tangles, thus providing information to aid in targeted AD therapeutics that could translate into positive clinical outcomes.


title: Sexual and temporal variation in New Zealand bellbird song repertoires

abstract: How song repertoires vary within species and change over time is well studied in male songbirds. However, variation in female song repertoires remains largely unstudied despite female song being much more common and complex than once assumed. We investigated the song syllable repertoire of the New Zealand bellbird (Anthornis melanura), a species where both sexes have complex but sexually dimorphic song. We compared songs at individual and population levels to investigate sex and temporal variation of syllable repertoires. We detected 96 syllable types in the population over four years, of which 58% were unique to males, 32% unique to females and 9% were shared between the sexes. The population syllable repertoire of both sexes changed substantially across years with similar turnover rates (Jaccards similarity coefficients; female 52.9-69.0%; male 58.6-73.7%). Furthermore, many syllable types, unique to each sex, varied in prevalence within the population across years. The syllable repertoire sizes of individuals were higher for males than females (13-32, n = 7 and 6-16, n = 8, respectively). Although these sample sizes were low, the temporal variation in syllable prevalence and turnover for individuals were similar to patterns at the population level. Overall, male and female bellbirds exhibited similarities in temporal patterns of yearly repertoire composition, with rapid changes in syllable prevalence, but females had fewer syllable types than males. We suggest that these similarities and differences are consistent with male and female song repertoires being driven by similar but not identical selection pressures.

category: animal behavior and cognition


title: Augmented Transcutaneous Stimulation Using an Injectable Electrode

abstract:
Minimally invasive neuromodulation technologies seek to marry the neural selectivity of implantable devices with the low-cost and non-invasive nature of transcutaneous electrical stimulation (TES). The Injectrode(R) is a needle-delivered electrode that is injected onto neural structures under image guidance. Power is then transcutaneously delivered to the Injectrode using surface electrodes. The Injectrode serves as a low-impedance conduit to guide current to the deep on-target nerve, reducing activation thresholds by an order of magnitude compared to using only surface stimulation electrodes. To minimize off-target recruitment of cutaneous fibers, the energy transfer efficiency from the surface electrodes to the Injectrode must be optimized.

TES energy is transferred to the Injectrode through both capacitive and resistive mechanisms. Electrostatic finite element models generally used in TES research consider only the resistive means of energy transfer by defining tissue conductivities. Here, we present an electroquasistatic model, taking into consideration both the conductivity and permittivity of tissue, to understand transcutaneous power delivery to the Injectrode. The model was validated with measurements taken from (n=4) swine cadavers. We used the validated model to investigate system and anatomic parameters that influence the coupling efficiency of the Injectrode energy delivery system. Our work suggests the relevance of electroquasistatic models to account for capacitive charge transfer mechanisms when studying TES, particularly when high-frequency voltage components are present, such as those used for voltage-controlled pulses and sinusoidal nerve blocks.

category: bioengineering


title: Characterization of new recombinant Immunoglobulins type-M binding to C1q by Biolayer Interferometry

abstract: The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs but due to their high oligomeric complexity, in vitro production, biochemical and biophysical characterizations are challenging. In the present study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (AUC, SEC-MALLS, Mass Photometry and Transmission Electron Microscopy). Each IgM oligomer has individual specific expression pattern and yield with different protein quality likely due to intrinsic IgM properties and patterning. Nevertheless, the purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. And more importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma.


title: Biophysical characterization of the oligomeric states of recombinant Immunoglobulins type-M and their C1q binding kinetics by Biolayer Interferometry


title: Repurposing CAR targets in solid tumors via a pan-cancer single-cell transcriptome analysis

abstract: Chimeric antigen receptor (CAR) T cell therapy is a powerful and promising tool for unleashing lasting antitumor immunity, which is currently studied in numerous blood and solid cancers. Here, mining single cell transcriptomics datasets of solid tumors, we survey many existing CAR targets, aiming to new solid tumor types in which these targets are differentially expressed in tumor cells and not on non-tumor cells within the tumor microenvironment, thus providing new repurposing opportunities. Quite surprisingly, we find that many existing solid cancer CAR targets have a better discriminatory power in uncharted cancer types than in the ones they have been developed or being tested for. Furthermore, we show that several existing liquid cancer CAR targets may also hold promise in specific solid tumors. Taken together, this analysis uncovers a large potential of repurposing current CARs that is currently yet untapped.


title: ReadZS detects developmentally regulated RNA processing programs in single cell RNA-seq and defines subpopulations independent of gene expression

abstract: Post-transcriptional regulation of RNA processing (RNAP), including splicing and alternative polyadenylation (APA), controls eukaryotic gene function. Conservative estimates based on bulk tissue studies conclude that at least 50% of mammalian genes undergo APA. Single-cell RNA sequencing (scRNA-seq) could enable a near complete estimate of the extent, function, and regulation of these and other forms of RNA processing. Yet, statistical methods to detect regulated RNAP are limited in their detection power because they suffer from reliance on (a) incomplete annotations of 3 untranslated regions (3 UTRs), (b) peak calling heuristics, (c) analysis based on measurements collapsed over all cells in a cell type (pseudobulking), or (d) APA-specific detection. Here, we introduce ReadZS, a computationally-efficient, and annotation-free statistical approach to identify regulated RNAP, including but not limited to APA, in single cells. ReadZS rediscovers and substantially extends the scope of known cell type-specific RNAP in the human lung and during human spermatogenesis. The unique single-cell resolution and statistical properties of ReadZS enable discovery of new evolutionarily conserved, developmentally regulated RNAP and subpopulations of lung-resident macrophages, homogenous by gene expression alone.


title: PEAK2VEC ENABLES INFERRENCE OF TRANSCRIPTIONAL REGULATION FROM ATAC-SEQ

abstract: Transcription factor (TF) binding sites in ATAC-seq are typically determined by footprint analysis. However, the performance of footprint analysis remains unsatisfying and most TFs do not exhibit footprint patterns. In this study, we modified the convolutional neural network to project sequences into an embedding space. Sequences with similar nucleotide patterns will stay close together in the embedding. The dimensionality of this embedding space represents binding specificities of various TFs. In the simulation experiment, peak2vec accurately distinguished the three TFs in the embedding space while conventional deep learning cannot. When applied to the ATAC-seq profiles of hepatitis carcinoma, peak2vec recovered multiple motifs curated in database, while significant portion of sequences corresponding to the TF are located at the promoter region of its regulated genes.


title: Revisiting colorectal cancer tumorigenesis with spatially-resolved gene expression profiling

abstract:
Early detection and treatment are paramount to the clinical outcome of patients with colorectal cancer (CRC). Deciphering the dynamic interactions that occur between epithelial cells and stromal cells during tumorigenesis requires in-depth analyses of early-stage CRC lesions in spatial context. Here we employed spatially-resolved gene expression profiling to dissect molecular processes that associate with malignant transformation in CRC. We provide the transcriptional landscapes of colorectal cancer tumorigenesis from healthy mucosa, through different degrees of dysplasia, to cancer. The complementary examination of epithelial and stromal fractions allowed us to define whether specific oncogenic processes involved cancer cells, stromal cells, or the tumor microenvironment as a whole. We identified several genes that were consistently deregulated during CRC onset that could serve as clinical biomarkers for early-stage CRC. Furthermore, we uncovered an essential role for the innate immune system during CRC tumorigenesis.

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title: Assessing extracellular vesicles from bovine mammary gland epithelial cells cultured in FBS-free medium

abstract:
AimMammary gland extracellular vesicles (EVs) are found in both human and livestock milk. Our knowledge of the role of EVs in the mammary gland development, breast cancer and mastitis derives mainly from in vitro cell culture models. However, a commonly shared limitation is the use of foetal bovine serum (FBS) as a supplement, which naturally contains EVs. For this reason, the purpose of the study is to establish a novel tool to investigate mammary gland EVs in vitro and in an FBS-free system.

MethodsPrimary bovine mammary epithelial cells (pbMECs) and a mammary gland alveolar epithelial cell line (MAC-T) were cultured in a chemically defined EV-free medium. To find a reliable EVs isolation protocol from a starting cell conditioned medium (10 mL), we compared eight different methodologies by combining ultracentrifugation (UC), chemical precipitation (CP), size exclusion chromatography (SEC), and ultrafiltration (UF).

ResultsThe medium formula sustained both pbMECs and MAC-T cell growth and did not alter MAC-T cell identity. Transmission electron microscopy revealed that we obtained EV-like particles in five out of eight protocols. The cleanest samples with the highest particles amount and detectable amounts of RNA were obtained by using UF-SEC-UC and UC-SEC-UC.

ConclusionOur chemically defined, EV-free medium sustains the growth of both pbMECs and MAC-T and allows the isolation of EVs that are free from any contamination by UF-SEC-UC and UC-SEC-UC. In conclusion, we propose a new culture system and EVs isolation protocols for further research on mammary epithelial EVs.

category: physiology


title: Female fruit flies cannot protect stored sperm from high temperature damage

abstract: Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. Here, we test whether females from two fruit fly species can protect stored sperm from a high temperature stress. We find that sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is destroying stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to destroy mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change.


title: Cost-effective, high-throughput phenotyping system for 3D reconstruction of fruit form

abstract: Reliable phenotyping methods that are simple to operate and inexpensive to deploy are critical for studying quantitative traits in plants. Traditional fruit shape phenotyping relies on human raters or 2D analyses to assess form, e.g., size and shape. Systems for 3D imaging using multi-view stereo have been implemented, but frequently rely on commercial software and/or specialized hardware, which can lead to limitations in accessibility and scalability. We present a complete system constructed of consumer-grade components for capturing, calibrating, and reconstructing the 3D form of small-to-moderate sized fruits and tubers. Data acquisition and image capture sessions are 9 seconds to capture 60 images. The initial prototype cost was $1600 USD. We measured accuracy by comparing reconstructed models of 3D printed ground truth objects to the original digital files of those same ground truth objects. The R2 between length of the primary, secondary, and tertiary axes, volume, and surface area of the ground-truth object and the reconstructed models was > 0.97 and root-mean square error (RMSE) was <3mm for objects without locally concave regions. Measurements from 1mm and 2mm resolution reconstructions were consistent (R2 > 0.99). Qualitative assessments were performed on 48 fruit and tubers, including 18 strawberries, 12 potatoes, 5 grapes, 7 peppers, and 4 Bosc and 2 red Anjou pears. Our proposed phenotyping system is fast, relatively low cost, and has demonstrated accuracy for certain shape classes, and could be used for the 3D analysis of fruit form.


title: Dominant Awn Inhibitor, which encodes an ALOG protein in sorghum, suppresses awn formation in rice

abstract: The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identified DOMINANT AWN INHIBITOR (DAI), which encodes the ALOG family protein on chromosome 3. DAI appeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed that DAI originated from duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that only DAI gained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that of DAIori. Interestingly, heterologous expression of DAI with its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found that DAI originated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.


title: DOMINANT AWN INHIBITOR encodes the ALOG protein originating from gene duplication and inhibits awn elongation by suppressing cell proliferation and elongation in sorghum


title: Structural basis for the absence of low-energy chlorophylls responsible for photoprotection from a primitive cyanobacterial PSI

abstract: Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-[A] resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. Since Gloeobacter is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.


title: Sound disrupts sleep-associated brain oscillations inrodents according to its meaning

abstract: Sleep is essential but poses a risk to the animal. Filtering acoustic information according to its relevance, a process generally known as sensory gating, is crucial during sleep to ensure a balance between rest and danger detection. The mechanisms of this sensory gating and its specificity are not understood. Here, we tested the effect that sounds of different meaning had on sleep-associated ongoing oscillations. We recorded EEG and EMG from mice during rapid-eye movement (REM) and non-REM (NREM) sleep while presenting sounds with or without behavioural relevance. We found that sound presentation per se, in the form of an unfamiliar neutral sound, elicited a weak or no change in the sleep-dependent EEG power during NREM and REM sleep. In contrast, the presentation of a sound previously conditioned in an aversive task, elicited a clear and fast decrease in the sleep-dependent EEG power during both sleep phases, suggesting a transition to lighter sleep without awakening. The observed changes generally weakened over training days and were not present in animals that failed to learn. Interestingly, the effect could be generalized to unfamiliar neutral sounds if presented following conditioned training, an effect that depended on sleep phase and sound type. The data demonstrate that sounds are differentially gated during sleep depending on their meaning and that this process is reflected in disruption of sleep-associated brain oscillations without an effect on behavioural arousal.


title: Natural sound characteristics explain perceptual categorization

abstract: As we interact with our surroundings, we encounter the same or similar objects from different perspectives and are compelled to generalize. For example, we recognize dog barks as a distinct class of sound, despite the variety of individual barks. While we have some understanding of how generalization is done along a single stimulus dimension, such as frequency or color, natural stimuli are identifiable by a combination of dimensions. To understand perception, measuring the interaction across stimulus dimensions is essential. For example, when identifying a sound, does our brain focus on a specific dimension or a combination, such as its frequency and duration? Furthermore, does the relative relevance of each dimension reflect its contribution to the natural sensory environment? Using a 2-dimension discrimination task for mice we tested untrained generalization across several pairs of auditory dimensions. We uncovered a perceptual hierarchy over the tested dimensions that was dominated by the sounds spectral composition. A model tuned to the predictability inherent in natural sounds best explained the behavioral results, suggesting that the perceptual hierarchy parallels the predictive content of natural sounds.


title: Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar

abstract: Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.


title: Distance estimation from monocular cues in an ethological visuomotor task

abstract: In natural contexts, sensory processing and motor output are closely coupled, which is reflected in the fact that many brain areas contain both sensory and movement signals. However, standard reductionist paradigms decouple sensory decisions from their natural motor consequences, and head-fixation prevents the natural sensory consequences of self-motion. In particular, movement through the environment provides a number of depth cues beyond stereo vision that are poorly understood. To study the integration of visual processing and motor output in a naturalistic task, we investigated distance estimation in freely moving mice. We found that mice use vision to accurately jump across a variable gap, thus directly coupling a visual computation to its corresponding ethological motor output. Monocular eyelid suture did not affect performance, thus mice can use cues that do not depend on binocular disparity and stereo vision. Under monocular conditions, mice performed more vertical head movements, consistent with the use of motion parallax cues, and optogenetic suppression of primary visual cortex impaired task performance. Together, these results show that mice can use monocular cues, relying on visual cortex, to accurately judge distance. Furthermore, this behavioral paradigm provides a foundation for studying how neural circuits convert sensory information into ethological motor output.


title: A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex

abstract: Identification of the structural connections between neurons is a prerequisite to understanding brain function. We developed a pipeline to systematically map brain-wide monosynaptic inputs to specific neuronal populations using Cre-driver mouse lines and the recombinant rabies tracing system. We first improved the rabies virus tracing strategy to accurately identify starter cells and to efficiently quantify presynaptic inputs. We then mapped brain-wide presynaptic inputs to different excitatory and inhibitory neuron subclasses in the primary visual cortex and seven higher visual areas. Our results reveal quantitative target-, layer- and cell-class-specific differences in the retrograde connectomes, despite similar global input patterns to different neuronal populations in the same anatomical area. The retrograde connectivity we define is consistent with the presence of the ventral and dorsal visual information processing streams and reveals further subnetworks within the dorsal stream. The hierarchical organization of the entire visual cortex can be derived from intracortical feedforward and feedback pathways mediated by upper- and lower-layer input neurons, respectively. This study expands our knowledge of the brain-wide inputs regulating visual areas and demonstrates that our improved rabies virus tracing strategy can be used to scale up the effort in dissecting connectivity of genetically defined cell populations in the whole mouse brain.


title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

abstract: Transition metals are toxic at high concentrations. The P1B-ATPase metal exporter CtpC/Rv3270 is required for resistance to zinc poisoning in the human pathogen Mycobacterium tuberculosis. Here, we discovered that zinc resistance also depends on the chaperone-like protein PacL1/Rv3269. PacL1 bound Zn2+, but unlike PacL1 and CtpC, the PacL1 metal-binding motif (MBM) was required only at high zinc concentrations. PacL1 co-localized with CtpC in dynamic microdomains within the mycobacterial plasma membrane. Microdomain formation did not require flotillins nor the PacL1 MBM. Instead, loss of the PacL1 Glutamine/Alanine repeats led to loss of CtpC and sensitivity to zinc. PacL1 and CtpC are within the same operon, and homologous PacL1-P1B-ATPase pairs are widely distributed within and across prokaryotes. PacL1 colocalized and functioned redundantly with PacL orthologs in Mycobacterium tuberculosis. Overall, our study suggests that PacL proteins are scaffolds that assemble P-ATPase-containing metal efflux platforms, a novel type of functional membrane microdomain that underlies bacterial resistance to metal poisoning.

category: microbiology


title: A genome-wide genetic screen uncovers novel determinants of human pigmentation

abstract:
The skin color is one of the most diverse human traits and is determined by the quantity, type and distribution of melanin. Here, we leverage light scattering properties of melanin to conduct a genome-wide CRISPR-Cas9 screen for novel regulators of melanogenesis. We identify functionally diverse genes converging on melanosome biogenesis, endosomal transport and transcriptional/posttranscriptional gene regulation, most of which represent novel associations with pigmentation. A survey of transcriptomes from diversely pigmented individuals reveals that the majority of genes discovered in our screen are upregulated in dark skin melanocytes, in agreement with their melanin-promoting function and potential contribution to skin color variation. This association is further buttressed by the significant skin color heritability enrichment in the vicinity of these genes. Taken together, our study presents a novel approach to assay pigmentation and uncovers a plethora of melanogenesis regulators, with broad implications for human variation, cell biology and medicine.

One Sentence SummaryGenetic screen uncovers genes involved in human melanogenesis, many of which are differentially expressed in individuals of diverse skin color.

category: cell biology


title: Nup358 regulates remodelling of ER-mitochondrial contact sites and autophagy

abstract: The contact sites between ER and mitochondria regulate several cellular processes including inter-organelle lipid transport, calcium homeostasis and autophagy. However, the mechanisms that regulate the dynamics and functions of these contact sites remain unresolved. We show that annulate lamellae (AL), a relatively unexplored subcellular structure representing subdomains of ER enriched with a subset of nucleoporins, are present at ER-mitochondria contact sites (ERMCS). Depletion of one of the AL-resident nucleoporins, Nup358, results in increased contacts between ER and mitochondria. Mechanistically, Nup358 modulates ERMCS dynamics by restricting mTORC2/Akt signalling. Our results suggest that growth factor-mediated remodelling of ERMCS depends on a reciprocal binding of Nup358 and mTOR to the ERMCS tethering complex consisting of VAPB and PTPIP51. Furthermore, Nup358 also interacts with IP3R, an ERMCS-enriched Ca2+ channel, and controls Ca2+ release from the ER. Consequently, depletion of Nup358 leads to elevated cytoplasmic Ca2+ and autophagy via activation of Ca2+/CaMKK2/AMPK axis. Our study thus uncovers a novel role for AL, particularly for Nup358, in regulating mTORC2-mediated ERMCS remodelling and Ca2+-directed autophagy, possibly via independent mechanisms.


title: S. pombe wtf genes use dual transcriptional regulation and selective protein exclusion from spores to cause meiotic drive

abstract:
Meiotic drivers bias gametogenesis to ensure their transmission into more than half the offspring of a heterozygote. In Schizosaccharomyces pombe, wtf meiotic drivers destroy the meiotic products (spores) that do not inherit the driver from a heterozygote, thereby reducing fertility. wtf drivers encode both a Wtfpoison protein and a Wtfantidote protein using alternative transcriptional start sites. Here, we analyze how the expression and localization of the Wtf proteins are regulated to achieve drive. We show that transcriptional timing and selective protein exclusion from developing spores ensure that all spores are exposed to Wtf4poison, but only the spores that inherit wtf4 receive a dose of Wtf4antidote sufficient for survival. In addition, we show that the Mei4 transcription factor, a master regulator of meiosis, controls the expression of the wtf4poison transcript. This dual transcriptional regulation, which includes the use of a critical meiotic transcription factor, likely complicates the universal suppression of wtf genes without concomitantly disrupting spore viability. We propose that these features contribute to the evolutionary success of the wtf drivers.

Author SummaryKiller meiotic drivers are one type of selfish DNA sequence. When only one copy of a killer meiotic driver is found in a genome, the driver is expected to be transmitted to only half of the gametes (e.g. eggs or sperm). Killer meiotic drivers, however, kill developing gametes that do not inherit them, giving the driver a transmission advantage into the next generation. The molecular mechanisms used by these killers are not well understood. In this work, we analyzed how one killer meiotic driver, wtf4 from fission yeast, ensures targeted gamete (spore) killing. Previous work showed that wtf meiotic drivers encode a poison protein that is transmitted to all spores and an antidote protein that rescues only spores that inherit the locus. Here, we show that different timing of the expression of the two proteins, combined with differential inclusion of the proteins in developing spores, both contribute to targeted spore killing. We also demonstrate that wtf4 exploits an essential gene expression pathway, making it difficult for the genome to prevent this locus from being expressed and killing. This extends our knowledge both of how these genetic parasites act and how they are equipped to evade host suppression mechanisms.


title: Characterizing Genetic Mechanisms Involved in Measuring Day-length in Drosophila melanogaster

abstract: Many organisms are known to regulate seasonal behaviors and physiological processes in response to day length changes through photoperiodism. Extreme changes in photoperiods have detrimental effects on human health, which can impair development and serve as the origin of adult diseases. Since the seminal work by Bunning in 1936, there are studies supporting the view that organisms can measure the day length through an endogenous 24-hour cellular circadian clock. However, the mechanisms involved in measuring seasonal or day-length changes are not understood. In the current study, we performed a genome-wide association study (GWAS) on photoperiodism using the Drosophila Genetic Reference Panel. The GWAS identified 32 candidate genes responsible for photoperiodic regulations. The knockout mutants of the top four candidate genes (Protein Kinase C delta (Pkcdelta), Glucuronyltransferase-P (GlcAT-P), Brain-specific homeobox (Bsh), and Diuretic hormone 31 Receptor (Dh31-R1)) were analyzed for their photoperiod and circadian period phenotypes. PKCdelta and GlcAT-P mutants show a significantly different photoperiod response compared to that of the wild type strain, and also had an altered circadian period phenotype. Further molecular characterization revealed that the mutant two independent mutant alleles of PKCdelta with a defective catalytic domain had distinct photoperiod responses. Taken these data together, we concluded that there is overlap between the circadian clock and photoperiodic regulations in Drosophila, and PKCdelta is a component that is involved in both circadian and photoperiodic regulations. By identifying novel molecular components of photoperiod, the current study provides new insights into the genetic mechanisms of determining the seasonal changes.


title: Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

abstract: Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete PRM2 incorporation, resulting in a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.


title: Comprehensive simulation and interpretation of single nucleotide substitutions in GJB2 reveals the genetic and phenotypic landscape of GJB2-related hearing loss

abstract: Genetic variants in the GJB2 gene are the most frequent causes of congenital and childhood hearing loss worldwide. In addition to nonsyndromic hearing loss, GJB2 pathogenic variants are also correlated with syndromic phenotypes, showing high genetic and phenotypic heterogeneity. To comprehensively delineate the genetic and phenotypic landscape of GJB2 variants, we interpreted and manually curated all the 2043 possible single-nucleotide substitution (SNS) coding variants in this gene following the hearing loss-specific ACMG/AMP guidelines. As a result, 61 (3.0%), 188 (9.2%), 1487 (72.8%), 301 (14.7%) and 6 (0.3%) variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign and benign, respectively. Interestingly, 54% (84/156) of pathogenic/likely pathogenic missense variants were not recorded in ClinVar. Further analysis showed that the second transmembrane domain (TM2) and the 310 helix are highly enriched for pathogenic missense variants. The N-terminal tail and the extracellular loop (E1) showed a high density of variants that are associated with syndromic or dominant nonsyndromic hearing loss. On the other hand, the intracellular loops (CL and CT) were extremely tolerant to variation. Based on this new information, we propose refinements of the guidelines for variant interpretation in GJB2. In summary, our study interpreted all possible SNS variants in the coding region of the GJB2 gene, characterized novel clinically significant (N = 249) and benign or likely benign (N = 307) in this gene, and revealed significant genotype-phenotype correlations at this common hearing loss locus. The interpretation of GJB2 SNS variants in the coding region provides a prototype for genes with similarly high genetic and phenotypic heterogeneity.


title: Destabilization of CAR T-cell treatment efficacy in the presence of dexamethasone

abstract:
Chimeric antigen receptor (CAR) T-cell therapy is potentially an effective targeted immunotherapy for glioblastoma, yet there is presently little known about the efficacy of CAR T-cell treatment when combined with the widely used anti-inflammatory and immunosuppressant glucocorticoid, dexamethasone. Here we present a mathematical model-based analysis of three patient-derived glioblastoma cell lines treated in vitro with CAR T-cells and dexamethasone. Advanced in vitro experimental cell killing assay technologies allow for highly resolved temporal dynamics of tumor cells treated with CAR T-cells and dexamethasone, making this a valuable model system for studying the rich dynamics of nonlinear biological processes with translational applications. We model the system as a nonautonomous, two-species predator-prey interaction of tumor cells and CAR T-cells, with explicit time-dependence in the clearance rate of dexamethasone. Using time as a bifurcation parameter, we show that (1) dexamethasone destabilizes coexistence equilibria between CAR T-cells and tumor cells in a dose-dependent manner and (2) as dexamethasone is cleared from the system, a stable coexistence equilibrium returns in the form of a Hopf bifurcation. With the model fit to experimental data, we demonstrate that high concentrations of dexamethasone antagonizes CAR T-cell efficacy by exhausting, or reducing the activity of CAR T-cells, and by promoting tumor cell growth. Finally, we identify a critical threshold in the ratio of CAR T-cell death to CAR T-cell proliferation rates that predicts eventual treatment success or failure that may be used to guide the dose and timing of CAR T-cell therapy in the presence of dexamethasone in patients.

Author summaryBioengineering and gene-editing technologies have paved the way for advance immunotherapies that can target patient-specific tumor cells. One of these therapies, chimeric antigen receptor (CAR) T-cell therapy has recently shown promise in treating glioblastoma, an aggressive brain cancer often with poor patient prognosis. Dexamethasone is a commonly prescribed anti-inflammatory medication due to the health complications of tumor associated swelling in the brain. However, the immunosuppressant effects of dexamethasone on the immunotherapeutic CAR T-cells are not well understood. To address this issue, we use mathematical modeling to study in vitro dynamics of dexamethasone and CAR T-cells in three patient-derived glioblastoma cell lines. We find that in each cell line studied there is a threshold of tolerable dexamethasone concentration. Below this threshold, CAR T-cells are successful at eliminating the cancer cells, while above this threshold, dexamethasone critically inhibits CAR T-cell efficacy. Our modeling suggests that in the presence of high dexamethasone reduced CAR T-cell efficacy, or increased exhaustion, can occur and result in CAR T-cell treatment failure.


title: Dose-dependent thresholds of dexamethasone destabilize CAR T-cell treatment efficacy


title: Chemotherapy-Induced Cachexia and Model-Informed Dosing to Preserve Lean Mass in Cancer Treatment

abstract:
Although chemotherapy is a standard treatment for cancer, it comes with significant side effects. In particular, certain agents can induce severe muscle loss, known as cachexia, worsening patient quality of life and treatment outcomes. 5-fluorouracil, an anti-cancer agent used to treat several cancers, has been shown to cause muscle loss. Experimental data indicates a non-linear dose-dependence for muscle loss in mice treated with daily or week-day schedules. We present a mathematical model of chemotherapy-induced muscle wasting that captures this non-linear dose-dependence. Area-under-the-curve metrics are proposed to quantify the treatments effects on lean mass and tumour control. Model simulations are used to explore alternate dosing schedules, aging effects, and morphine use in chemotherapy treatment with the aim of better protecting lean mass while actively targeting the tumour, ultimately leading to improved personalization of treatment planning and improved patient quality of life.

Author SummaryIn this paper we present a novel mathematical model for muscle loss due to cancer chemotherapy treatment. Loss of muscle mass relates to increased drug toxicity and side-effects, and to decreased patient quality of life and survival rates. With our model, we examine the therapeutic efficacy of various dosing schedules with the aim of controlling a growing tumour while also preserving lean mass. Preservation of body composition, in addition to consideration of inflammation and immune interactions, the gut microbiome, and other systemic health measures, may lead to improved patient-specific treatment plans that improve patient quality of life.


title: Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation under Ionic Liquid Stress

abstract: Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs. absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.

category: systems biology


title: Integrated Regulation of PKA by Fast and Slow Neurotransmission in the Nucleus Accumbens Controls Plasticity and Stress Responses

abstract: Cortical glutamate and midbrain dopamine neurotransmission converge to mediate striatum-dependent behaviors, while maladaptations in striatal circuitry contribute to mental disorders. Here we uncover a molecular mechanism by which glutamatergic and dopaminergic signaling integrate to regulate cAMP-dependent protein kinase (PKA) via phosphorylation of the PKA regulatory subunit, RII{beta}. We find that glutamate-dependent reduction in Cdk5-dependent RII{beta} phosphorylation alters the PKA holoenzyme auto-inhibitory state to increase PKA signaling in response to dopamine. Disruption of RII{beta} phosphorylation by Cdk5, consequently, enhances cortico-ventral striatal synaptic plasticity. Acute and chronic stress in rats inversely modulate RII{beta} phosphorylation and ventral striatal infusion of a small interfering peptide that selectively targets RII{beta} regulation by Cdk5 improves behavioral response to stress. This new signaling mechanism integrating ventral striatal glutamate and dopamine neurotransmission is likely important to brain function, may contribute to neuropsychiatric conditions, and serves as a possible target for the development of novel therapeutics for stress-related disorders.


title: Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

abstract: We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+ concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]i rise, which was reversible upon TG2 removal and was dependent on Ca2+ influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarisation and activation of inward currents, due to the opening of L-type-VOCCs and to Na+/Ca2+ -exchanger (NCX) operation in the reverse mode, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+ influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]i through release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation.


title: Regulation of the Evolutionarily Conserved Muscle Myofibrillar Matrix by Cell Type Dependent and Independent Mechanisms

abstract: Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. We recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. Here, we demonstrate the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, we find that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and we show that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, we demonstrate that neurochondrin knockdown results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms.


title: In Vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras

abstract:
Generation of bone marrow (BM) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce all BM hematopoietic cell lineages from mouse ESCs in a rat. Based on FACS analysis and single-cell RNA sequencing, mouse ESCs differentiated into hematopoietic progenitor cells and multiple hematopoietic cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Transplantation of ESC-derived BM cells from mouse-rat chimeras rescued lethally-irradiated syngeneic mice and resulted in long-term contribution of donor cells to hematopoietic cell lineages. Altogether, a fully functional bone marrow was generated from mouse ESCs using rat embryos as "bioreactors".

KEY POINTS- We used blastocyst complementation to simultaneously produce all bone marrow hematopoietic cell lineages from mouse ESCs in a rat.

- ESC-derived cells from mouse-rat chimeras were fully functional and exhibited normal gene expression signatures and cell surface markers.


title: Bsh coordinates neuronal fate specification with synaptic connectivity

abstract: It is widely accepted that neuronal fate is initially determined by spatial and temporal cues acting in progenitors, followed by transcription factors (TFs) that act in post-mitotic neurons to specify their functional identity (e.g. ion channels, cell surface molecules, and neurotransmitters). It remains unclear, however, whether a single TF can coordinately regulate both steps. The five lamina neurons (L1-L5) in the Drosophila visual system, are an ideal model for addressing this question. Here we show that the homeodomain TF Brain-specific homeobox (Bsh) is expressed in a subset of lamina precursor cells (LPCs) where it specifies L4 and L5 fate, and suppresses homeodomain TF Zfh1 to prevent L1 and L3 fate. Subsequently, in L4 neurons, Bsh initiates a feed forward loop with another homeodomain TF Apterous (Ap) to drive recognition molecule DIP-{beta} expression, which is required for precise L4 synaptic connectivity. We conclude that a single homeodomain TF expressed in both precursors and neurons can coordinately generate neuronal fate and synaptic connectivity, thereby linking these two developmental events. Furthermore, our results suggest that acquiring LPC expression of a single TF, Bsh, may be sufficient to drive the evolution of increased brain complexity.


title: Transcriptional fidelity enhances cancer cell line selection in pediatric cancers

abstract: Multi-omic technologies have allowed for comprehensive profiling of patient-derived tumor samples and the cell lines that are intended to model them. Yet, our understanding of how cancer cell lines reflect native pediatric cancers in the age of molecular subclassification remains unclear and represents a clinical unmet need. Here we use Treehouse public data to provide an RNA-seq driven analysis of 799 cancer cell lines, focusing on how well they correlate to 1,655 pediatric tumor samples spanning 12 tumor types. For each tumor type we present a ranked list of the most representative cell lines based on correlation of their transcriptomic profiles to those of the tumor. We found that most (8/12) tumor types best correlated to a cell line of the closest matched disease type. We furthermore showed that inferred molecular subtype differences in medulloblastoma significantly impacted correlation between medulloblastoma tumor samples and cell lines. Our results are available as an interactive web application to help researchers select cancer cell lines that more faithfully recapitulate pediatric cancer.


title: Enhancers of host immune tolerance to bacterial infection discovered using linked computational and experimental approaches

abstract: Current therapeutic strategies against bacterial infections focus on reduction of pathogen load through antibiotics; however, stimulation of host tolerance to infection might offer an alternative approach. Here we used computational transcriptomics and a Xenopus embryo infection model to rapidly discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus embryos exhibit natural tolerance to A. baumanii, K. pneumoniae, S. aureus, and S. pneumoniae bacteria, whereas A. hydrophila and P. aeruginosa produce infection that leads to death. Transcriptional profiling led to definition of a 20-gene signature that allows for discrimination between tolerant and susceptible states, as well as identification of active and passive tolerance responses based on the degree of engagement of gene transcription modulation. Upregulation of metal ion transport and hypoxia pathways reminiscent of responses observed in primate and mouse infection models were identified as tolerance mediators, and drug screening in the susceptible A. hydrophila infection model confirmed that a metal chelator (deferoxamine) and HIF-1 agonist (1,4-DPCA) increase embryo survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with multi-omics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infection.

category: immunology


title: Bioactive isoprenoids guide migrating germ cells to the embryonic gonad

abstract: Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in insects, impact the germline lifecycle from meiosis to gametogenesis. Emerging evidence suggests that these bioactive isoprenoids broadly influence embryonic reproductive development, though the precise functions remain unclear. Here, we investigated the role of bioactive isoprenoids in early germ cell development using JHs in Drosophila as a model. With a newly generated in vivo reporter, we find that JH signaling is active in the vicinity of germ cells as they migrate to colonize the somatic gonad. Through a combination of in vivo and in vitro assays, we find that JHs are both necessary and sufficient for primordial germ cell migration through mechanisms independent of canonical nuclear receptor-mediated transcription. These findings reveal that JH is present during Drosophila embryogenesis and that bioactive isoprenoids impact germ cell development earlier than previously appreciated. Interestingly, we find that like JH in Drosophila, RA is sufficient for murine germ cell migration in vitro, suggesting that the impact of bioactive isoprenoids on embryonic germ cell development may be broadly conserved.


title: Dual TBX5-Lineage and MYL2 Reporter System For Identification of Left Ventricular Cardiomyocytes During Human Induced Pluripotent Stem Cell Differentiation

abstract: During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF-progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.


title: Combined Lineage Tracing and scRNA-seq Reveals Unexpected First Heart Field Predominance of Human iPSC Differentiation