title: Non-alcoholic components of Pelinkovac, a Croatian wormwood-based strong liquor, counteract the inhibitory effect of high ethanol concentration on catalase in vitro

abstract: Antioxidant enzyme catalase protects the cells against alcohol-induced oxidative stress by scavenging free radicals and metabolizing alcohol. Concentrations of ethanol present in alcoholic beverages can inhibit catalase and foster oxidative stress and alcohol-induced injury. Non-alcoholic components of pelinkovac counteract the inhibitory effects of high ethanol concentration and acidic pH on catalase in vitro.


title: Rnf149-related is an FGF/MAPK-independent regulator of pharyngeal muscle fate specification

abstract: During embryonic development, cell fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors to both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study the cardiopharyngeal fate specification with cellular resolution, as only 2 pairs of cardiopharyngeal multipotent progenitors give rise to the heart and to pharyngeal muscles (aka atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented asymmetric divisions. Here, we identify the primed gene Rnf149-related (Rnf149-r), which becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss knock-out of Rnf149-r function impairs atrial siphon muscle morphogenesis, and down-regulates Tbx1/10 and Ebf, two key determinants of the pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of loss of FGF-MAPK signaling in the cardiopharyngeal lineage, and integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations identified a significant overlap between FGF-MAPK and Rnf149-r targets. However, functional interaction assays suggested the Rnf149-r does not directly modulate the activity of the FGF-MAPK-Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF-MAPK signaling on shared targets, as well as on FGF-MAPK-independent targets through (a) separate pathway(s).

category: developmental biology


title: Homologous BHLH transcription factors induce distinct deformations of torsionally-stressed DNA: a potential transcription regulation mechanism.

abstract: Changing torsional restraints on DNA is essential for the regulation of transcription. Torsional stress, introduced by RNA polymerase, can propagate along chromatin facilitating topological transitions and modulating the specific binding of transcription factors (TFs) to DNA. Despite the importance, the mechanistic details on how torsional stress impacts the TFs-DNA complexation remain scarce. Herein we address the impact of torsional stress on DNA complexation with homologous human basic-helix-loop-helix (BHLH) hetero- and homodimers: MycMax, MadMax, and MaxMax. The three TF dimers exhibit specificity towards the same DNA consensus sequences, the E-box response element, while regulating different transcriptional pathways. Using microseconds-long atomistic molecular dynamics simulations together with the torsional restraint that controls DNA total helical twist, we gradually over- and underwind naked and complexed DNA to a maximum of {+/-}5{degrees}/b.p. step. We observe that the binding of the BHLH dimers results in a similar increase in DNA torsional rigidity. However, under torsional stress the BHLH dimers induce distinct DNA deformations, characterised by changes in DNA grooves geometry and a significant asymmetric DNA bending. Supported by bioinformatics analyses, our data suggest that torsional stress may contribute to the execution of differential transcriptional programs of the homologous TFs by modulating their collaborative interactions.

category: biochemistry


title: Thermal characterization of the multimeric bacteriophage endolysin PlyC

abstract: Bacteriophage endolysins degrade the bacterial peptidoglycan and are considered enzymatic alternatives to small molecule antibiotics. In particular, the multimeric streptococcal endolysin PlyC has appealing antibacterial properties. However, a comprehensive thermal analysis of PlyC is lacking, which is necessary for evaluating long-term stability and downstream therapeutic potential. Biochemical and kinetic-based methods were used in combination with differential scanning calorimetry to investigate the structural, kinetic and thermodynamic stability of PlyC and its various subunits and domains. The PlyC holoenzyme structure is irreversibly compromised due to partial unfolding and aggregation at 46{o}C. Unfolding of the catalytic subunit, PlyCA, instigates this event, resulting in the kinetic inactivation of the endolysin. In contrast to PlyCA, the PlyCB octamer (the cell wall binding domain) is thermostable, denaturing at [~]75{o}C. Isolation of PlyCA or PlyCB alone altered their thermal properties. Contrary to the holoenzyme, PlyCA alone unfolds uncooperatively and is thermodynamically destabilized whereas the PlyCB octamer reversibly dissociates into monomers and forms an intermediate state at 74{o}C in phosphate buffered saline, with each subunit subsequently denaturing at 92{o}C. Adding folded PlyCA to an intermediate state PlyCB, followed by cooling, allowed for in vitro reconstitution of the active holoenzyme.

category: biophysics


title: Translational buffering by ribosome stalling in upstream open reading frames

abstract: Upstream open reading frames (uORFs) are present in over half of all human mRNAs. uORFs can potently regulate the translation of downstream open reading frames by several mechanisms: siphoning away scanning ribo-somes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. Here, we performed systematic measurements on the uORF-containing 5 UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF translation when ribosome loading onto the mRNA is reduced. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. We identify several human uORFs that repress main ORF translation via a similar terminal diproline motif. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions.

category: molecular biology


title: Fluid-structure interaction analysis of eccentricity and leaflet rigidity on thrombosis biomarkers in bioprosthetic aortic valve replacements

abstract: This work introduces the first 2-way fluid-structure interaction (FSI) computational model to study the effect of aortic annulus eccentricity on the performance and thrombogenic risk of cardiac bioprostheses. The model predicts that increasing eccentricities yield lower geometric orifice areas (GOAs) and higher normalized transvalvular pressure gradients (TPGs) for healthy cardiac outputs during systole, agreeing with in vitro experiments. Regions with peak values of residence time and shear rate are observed to grow with eccentricity in the sinus of Valsalva, indicating an elevated risk of thrombus formation for eccentric configurations. In addition, the computational model is used to analyze the effect of varying leaflet rigidity on both performance, thrombogenic and calcification risks with applications to tissue-engineered prostheses, observing an increase in systolic and diastolic TPGs, and decrease in systolic GOA, which translates to decreased valve performance for more rigid leaflets. An increased thrombogenic risk is detected for the most rigid valves. Peak solid stresses are also analyzed, and observed to increase with rigidity, elevating risk of valve calcification and structural failure. The immersed FSI method was implemented in a high-performance computing multi-physics simulation software, and validated against a well known FSI benchmark. The aortic valve bioprosthesis model is qualitatively contrasted against experimental data, showing good agreement in closed and open states. To the authors knowledge this is the first computational FSI model to study the effect of eccentricity or leaflet rigidity on thrombogenic biomarkers, providing a novel tool to aid device manufacturers and clinical practitioners.

category: bioengineering


title: Resolving Protein Conformational Plasticity and Substrate Binding Through the Lens of Machine-Learning

abstract: A long-standing target in elucidating the biomolecular recognition process is the identification of binding-competent conformations of the receptor protein. However, protein conformational plasticity and the stochastic nature of the recognition processes often preclude the assignment of a specific protein conformation to an individual ligand-bound pose. In particular, we consider multi-microsecond long Molecular dynamics simulation trajectories of ligand recognition process in solvent-inaccessible cavity of two archtypal systems: L99A mutant of T4 Lysozyme and Cytochrome P450. We first show that if the substrate-recognition occurs via long-lived intermediate, the protein conformations can be automatically classified into substrate-bound and unbound state through an unsupervised dimensionality reduction technique. On the contrary, if the recognition process is mediated by selection of transient protein conformation by the ligand, a clear correspondence between protein conformation and binding-competent macrostates can only be established via a combination of supervised machine learning (ML) and unsupervised dimension reduction approach. In such scenario, we demonstrate that an a priori random forest based supervised classification of the simulated trajectories recognition process would help characterize key amino-acid residue-pairs of the protein that are deemed sensitive for ligand binding. A subsequent unsupervised dimensional reduction via time-lagged independent component analysis of the selected residue-pairs would delineate a conformational landscape of protein which is able to demarcate ligand-bound pose from the unbound ones. As a key breakthrough, the ML-based protocol would identify distal protein locations which would be allosterically important for ligand binding and characterise their roles in recognition pathways.


title: Differences in pathways contributing to thyroid hormone effects on postnatal cartilage calcification versus secondary ossification center development

abstract: The proximal and distal femur epiphysis of mice are both weight bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH) in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in Thyroid stimulating hormone receptor (Tshr-/-) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo, and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization (Bsp, Ocn, Dmp1, Opn, and Alp). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.

category: physiology


title: Impact of adolescent intermittent ethanol exposure on interneurons and their surrounding perineuronal nets in adulthood.

abstract:
BackgroundBinge alcohol exposure during adolescence results in long-lasting alterations in brain and behavior. For example, adolescent intermittent ethanol (AIE) exposure in rodents results in long-term loss of functional connectivity among prefrontal cortex (PFC) and striatal regions as well as a variety of neurochemical, molecular, and epigenetic alterations. Interneurons in the PFC and striatum play critical roles in behavioral flexibility and functional connectivity. For example, parvalbumin (PV) interneurons are known to contribute to neural synchrony, and cholinergic interneurons contribute to strategy selection. Furthermore, extracellular perineuronal nets (PNNs) surround some interneurons, particularly PV+ interneurons, to further regulate cellular plasticity. The effect of AIE exposure on expression of these markers within the PFC is not well understood.

MethodsThe present study tested the hypothesis that AIE exposure reduces expression of PV+ and ChAT+ interneurons in the adult PFC and striatum and increases related expression of PNNs (marked by binding of Wisteria Floribunda agglutinin lectin; WFA) in adulthood. Male rats were exposed to AIE (5 g/kg/day, 2-days-on/2-days-off, i.g., P25-P54) or water (CON), and brain tissue was harvested in adulthood (> P80). Immunohistochemistry and co-immunofluorescence were used to assess expression of ChAT, PV, and WFA labeling within the adult PFC and striatum following AIE exposure.

ResultsChAT and PV interneuron numbers in the striatum and PFC were unchanged after AIE exposure. However, WFA labeling in the PFC of AIE-exposed rats was increased compared to CON rats. Moreover, significantly more PV neurons were surrounded by WFA labeling in AIE-exposed subjects relative to controls in both PFC subregions assessed: the orbitofrontal cortex (CON = 34%; AIE = 40%) and the medial PFC (CON = 10%; AIE = 14%).

ConclusionsThese findings indicate that while PV interneuron expression in the adult PFC and striatum is unaltered following AIE exposure, PNNs surrounding these neurons (indicated by extracellular WFA binding) are increased. This increase in PNNs may restrict plasticity of the ensheathed neurons, thus contributing to impaired microcircuitry in frontostriatal connectivity and related behavioral impairments.

category: neuroscience


title: Loss-of-function of OTUD7A in the schizophrenia-associated 15q13.3 deletion impairs synapse development and function in human neurons

abstract: Identifying causative gene(s) within disease-associated large genomic regions of copy number variants (CNVs) is challenging. Here, by targeted sequencing of genes within schizophrenia (SZ)-associated CNVs in 1,779 SZ cases and 1,418 controls, we identified three rare putative loss-of-function (LoF) mutations in OTU deubiquitinase 7A (OTUD7A) within the 15q13.3 deletion in cases, but none in controls. To tie OTUD7A LoF with any SZ-relevant cellular phenotypes, we modeled the OTUD7A LoF mutation, rs757148409, in human induced pluripotent stem cell (hiPSC)-derived induced excitatory neurons (iNs) by CRISPR/Cas9 engineering. The mutant iNs showed a [~]50% decrease in OTUD7A expression without undergoing nonsense-mediated mRNA decay. The mutant iNs also exhibited marked reduction of dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity. Congruent with the neuronal phenotypes in mutant iNs, our transcriptomic analysis showed that the set of OTUD7A LoF-downregulated genes was enriched for those relating to synapse development and function, and was associated with SZ and other neuropsychiatric disorders. These results suggest that OTUD7A LoF impairs synapse development and neuronal function in human neurons, providing mechanistic insight into the possible role of OTUD7A in driving neuropsychiatric phenotypes associated with the 15q13.3 deletion.


title: Characterization of ultrapotent chemogenetic ligands for research applications in non-human primates

abstract: Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of "Pharmacologically Selective Actuator Modules" (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent "Pharmacologically Selective Effector Molecules" (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in in vitro studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus macaques received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817(0.064 mg/kg) or uPSEM792 (0.87 mg/kg) and plasma and CSF samples were collected over the course of 48 hours. Both compounds exhibited good brain penetrance, relatively slow washout and negligible conversion to potential metabolites - varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.


title: Extracting abundance information from DNA-based data

abstract: The accurate extraction of species-abundance information from DNA-based data (metabarcoding, metagenomics) could contribute usefully to diet analysis and food-web reconstruction, the inference of species interactions, the modelling of population dynamics and species distributions, the biomonitoring of environmental state and change, and the inference of false positives and negatives. However, capture bias, capture noise, species pipeline biases, and pipeline noise all combine to inject error into DNA-based datasets. We focus on methods for correcting species pipeline biases and pipeline noise, as the first two errors are addressed extensively in the ecological literature. To extract abundance information, it is useful to distinguish two concepts. (1) Across-species quantification describes relative species abundances within one sample. (2) Within-species quantification describes how the abundance of each individual species varies from sample to sample, as in a time series, an environmental gradient, or experimental treatments. Firstly, we review methods to remove species pipeline biases and pipeline noise. Secondly, we demonstrate experimentally (with detailed protocol) how to use a  DNA spike-in to remove pipeline noise and recover within-species abundance information. We also introduce a model-based estimator that can be employed on datasets without a physical spike-in to approximately estimate and correct for pipeline noise.

category: ecology


title: GraphChainer: Co-linear Chaining for Accurate Alignment of Long Reads to Variation Graphs

abstract:
Aligning reads to a variation graph is a standard task in pangenomics, with downstream applications in e.g., improving variant calling. While the vg toolkit (Garrison et al., Nature Biotechnology, 2018) is a popular aligner of short reads, GraphAligner (Rautiainen and Marschall, Genome Biology, 2020) is the state-of-the-art aligner of long reads. GraphAligner works by finding candidate read occurrences based on individually extending the best seeds of the read in the variation graph. However, a more principled approach recognized in the community is to co-linearly chain multiple seeds. We present a new algorithm to co-linearly chain a set of seeds in an acyclic variation graph, together with the first efficient implementation of such a co-linear chaining algorithm into a new aligner of long reads to variation graphs, GraphChainer. Compared to GraphAligner, at a normalized edit distance threshold of 40%, it aligns 9% to 12% more reads, and 15% to 19% more total read length, on real PacBio reads from human chromosomes 1 and 22. On both simulated and real data, GraphChainer aligns between 97% and 99% of all reads, and of total read length. At the more stringent normalized edit distance threshold of 30%, GraphChainer aligns up to 29% more total real read length than GraphAligner.

GraphChainer is freely available at https://github.com/algbio/GraphChainer

category: bioinformatics


title: Assessing and assuring interoperability of a genomics file format

abstract:
BackgroundBioinformatics software tools operate largely through the use of specialized genomics file formats. Often these formats lack formal specification, and only rarely do the creators of these tools robustly test them for correct handling of input and output. This causes problems in interoperability between different tools that, at best, wastes time and frustrates users. At worst, interoperability issues could lead to undetected errors in scientific results.

MethodsWe sought (1) to assess the interoperability of a wide range of bioinformatics software using a shared genomics file format and (2) to provide a simple, reproducible method for enhancing inter-operability. As a focus, we selected the popular Browser Extensible Data (BED) file format for genomic interval data. Based on the file formats original documentation, we created a formal specification. We developed a new verification system, Acidbio (https://github.com/hoffmangroup/acidbio), which tests for correct behavior in bioinformatics software packages. We crafted tests to unify correct behavior when tools encounter various edge cases--potentially unexpected inputs that exemplify the limits of the format. To analyze the performance of existing software, we tested the input validation of 80 Bioconda packages that parsed the BED format. We also used a fuzzing approach to automatically perform additional testing.

ResultsOf 80 software packages examined, 75 achieved less than 70% correctness on our test suite. We categorized multiple root causes for the poor performance of different types of software. Fuzzing detected other errors that the manually designed test suite could not. We also created a badge system that developers can use to indicate more precisely which BED variants their software accepts and to advertise the softwares performance on the test suite.

DiscussionAcidbio makes it easy to assess interoperability of software using the BED format, and therefore to identify areas for improvement in individual software packages. Applying our approach to other file formats would increase the reliability of bioinformatics software and data.


title: Comparing transmission reconstruction models with Mycobacterium tuberculosis whole genome sequence data

abstract: Pathogen genomic epidemiology is now routinely used worldwide to interrogate infectious disease dynamics. Multiple computational tools that reconstruct transmission networks by coupling genomic data with epidemiological modelling have been developed. The resulting inferences are often used to inform outbreak investigations, yet to date, the performance of these transmission reconstruction tools has not been compared specifically for tuberculosis, a disease process with complex epidemiology that includes variable latency periods and within-host heterogeneity. Here, we carried out a systematic comparison of seven publicly available transmission reconstruction tools, evaluating their accuracy in predicting transmission events in both simulated and real-world Mycobacterium tuberculosis outbreaks. No tool was able to fully resolve transmission networks, though both the single-tree and multi-tree input implementations of TransPhylo identified the most epidemiologically supported transmission events and the fewest false positive links. We observed a high degree of variability in the transmission networks inferred by each approach. Our findings may inform an end-users choice of tools in future tuberculosis transmission analyses and underscore the need for caution when interpreting transmission networks produced using probabilistic approaches.


title: CHDbase: A Comprehensive Knowledgebase for Congenital Heart Disease-related Genes and Clinical Manifestations

abstract: Congenital heart disease (CHD) is the most common cause of major birth defects, with a prevalence of 1%. Although an increasing number of studies reporting the etiology of CHD, the findings scattered throughout the literature are difficult to retrieve and utilize in research and clinical practice. We therefore developed CHDbase, an evidence-based knowledgebase with CHD-related genes and clinical manifestations manually curated from 1114 publications, linking 1124 susceptibility genes and 3591 variations to more than 300 CHD types and related syndromes. Metadata such as the information of each publication and the selected population and samples, the strategy of studies, and the major findings of study were integrated with each item of research record. We also integrated functional annotations through parsing ~50 databases/tools to facilitate the interpretation of these genes and variations in disease pathogenicity. We further prioritized the significance of these CHD-related genes with a gene interaction network approach, and extracted a core CHD sub-network with 163 genes. The clear genetic landscape of CHD enables the phenotype classification based on the shared genetic origin. Overall, CHDbase provides a comprehensive and freely available resource to study CHD susceptibility, supporting a wide range of users in the scientific and medical communities. CHDbase is accessible at http://chddb.fwgenetics.org/.

category: genetics


title: Common neural functions during children's naturalistic and controlled laboratory mathematics learning

abstract: A major goal of human neuroscience is to understand how the brain functions in the real world, and to measure neural processes under conditions that are ecologically valid. A critical step toward this goal is understanding how brain activity during naturalistic tasks that mimic the real world, relates to brain activity in more traditional laboratory tasks. In the present study, we used intersubject correlations to locate reliable stimulus-driven cerebral processes among children and adults in a naturalistic video lesson and a laboratory forced-choice task that shared the same arithmetic concept. We show that relative to a control condition with grammatical content, naturalistic and laboratory arithmetic tasks evoked overlapping activation within brain regions previously associated with math semantics. The regions of specific functional overlap between the naturalistic mathematics lesson and laboratory mathematics task included bilateral intraparietal cortex, which confirms that this region processes mathematical content independently of differences in task mode. These findings suggest that regions of the intraparietal cortex process mathematical content when children are learning about mathematics in the real world.


title: Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor

abstract: Thyroid hormones are vital to growth and metabolism. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR). Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves disease. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright conformation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody selects a similar upright conformation of the extracellular domain. Conformational changes in the extracellular domain are transduced to the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR ECD relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to G protein-coupled receptors with large extracellular domains.


title: Wnt/PCP signaling mediates breast cancer metastasis by promoting pro-invasive protrusion formation in collectively motile leader cells

abstract: As evidence supporting essential roles for collective cell migration in carcinoma metastasis continues to accumulate, a better understanding of the underlying cellular and molecular mechanisms will be critical to translating these findings to the treatment of advanced cancers. Here we report that Wnt/PCP, a non-canonical Wnt signaling pathway, mediates breast cancer collective migration and metastasis. We observe that mammary gland-specific knockout of Vangl2, a tetraspanin-like scaffolding protein required for Wnt5a-induced signaling and motility in cultured breast cancer cell lines, results in a striking decrease in metastatic efficiency but not primary tumor growth in the MMTV-NDL transgenic mouse model of HER2-positive breast cancer. We also observe that expression levels of core Wnt/PCP components Wnt5a, Vangl1 and Vangl2 are selectively elevated in K14-positive leader cells relative to follower cells within a collectively migrating cohort, and that Vangl2 expression selectively promotes RhoA activation in leading edge cells. Moreover, Vangl expression drives collective migration in three-dimensional ex vivo tumor organoids, and Vangl protein specifically accumulates within pro-migratory filamentous actin-rich protrusions of leader cells. Together, our observations point to a model whereby Wnt/PCP upregulation facilitates breast tumor collective cell motility by selectively augmenting the formation pro-migratory protrusions within leader cells.

category: cancer biology


title: Busy and confused? High risk of missed alerts in the cockpit: an electrophysiological study

abstract: Of evolutionary importance, the ability to react to unexpected auditory stimuli remains critical today, especially in settings such as aircraft cockpits or air traffic control towers, characterized by high mental and auditory loads. Evidences show that both factors can negatively impact auditory attention and prevent appropriate reactions in hazardous situations. In the present study, sixty participants performed a simulated aviation task, varying in terms of mental load (no, low, high mental load), that was embedded with a concurrent tone detection paradigm, in which auditory load was manipulated by the number of different tones (1, 2 or 3). We measured both detection performance (miss, false alarm) and brain activity (event-related potentials) related to the target tone. Our results showed that both mental and auditory loads affected tone detection performance. Importantly, their combined effects had a massive impact on the percentage of missed target tones. While, in the no mental load condition, miss rate was very low with 1 (0.53%) and 2 tones (1.11%), it increased drastically with 3 tones (24.44%), and this effect was accentuated as mental load increased, yielding to the higher miss rate in the 3-tone paradigm under high mental load conditions (68.64%). Increased mental load, auditory load, and miss rate, were all associated with disrupted brain response to the target tone as showed by reductions of the P3b amplitude. In sum, our results highlight the importance of balancing mental and auditory loads to maintain or improve efficient reactions to alarms in complex environment.


title: Raman Signal Denoising Using Fully Convolutional Encoder Decoder Network

abstract: Raman spectroscopy is a vibrational method that gives molecular information rapidly and non-invasively. Despite its advantages, the weak intensity of Raman spectroscopy leads to low-quality signals, particularly with tissue samples. The requirement of high exposure times makes Raman a time-consuming process and diminishes its non-invasive property while studying living tissues. Novel denoising techniques using convolutional neural networks (CNN) have achieved remarkable results in image processing. Here, we propose a similar approach for noise reduction for the Raman spectra acquired with 10x lower exposure times. In this work, we developed fully convolutional encoder-decoder architecture (FCED) and trained them with noisy Raman signals. The results demonstrate that our model is superior (p-value < 0.0001) to the conventional denoising techniques such as the Savitzky-Golay filter and wavelet denoising. Improvement in the signal-to-noise ratio values ranges from 20% to 80%, depending on the initial signal-to-noise ratio. Thus, we proved that tissue analysis could be done in a shorter time without any need for instrumental enhancement.


title: Raman Signal Denoising using Fully Convolutional Encoder Decoder Network


title: Features of smaller ribosomes in Candidate Phyla Radiation (CPR) bacteria revealed with a molecular evolutionary analysis

abstract: The Candidate Phyla Radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and often lack ribosomal proteins L1, L9, and/or L30, which are basically ubiquitous in ordinary (non-CPR) bacteria. Here, we comprehensively analyzed the genomic information of CPR bacteria and identified their unique properties. In the distribution of protein lengths in CPR bacteria, the peak was at around 100-150 amino acids, whereas the position of the peak varies in the range of 100-300 amino acids in free-living non-CPR bacteria, and at around 100-200 amino acids in most symbiotic non-CPR bacteria. These results show that CPR bacteria have smaller proteins on average, like symbiotic non-CPR bacteria. We found that ribosomal proteins L28, L29, L32, and L33 are also deleted in CPR bacteria, in a lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins in CPR differ, in part, from those of non-CPR bacteria, with missing regions or specific added region. We also found that several regions of the 16S, 23S, and 5S rRNAs are lacking in CPR bacteria and that the total predicted length of the three rRNAs in CPR bacteria is smaller than that in non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than in free-living non-CPR bacteria, with simplified surface structures.


title: Hexosamine pathway activation improves memory but does not extend lifespan in mice.

abstract: Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension in various species. Thus, there is considerable interest in the potential health benefits of dietary supplementation with glucosamine or other HBP metabolites in people. The HBP is a sensor for energy availability and its activation has been implicated in tumor progression and diabetes in higher organisms. As the activation of the HBP has been linked to longevity in lower animals, it is imperative to explore the long-term effects of chronic HBP activation in mammals, which has not been examined so far. To address this issue, we activated the HBP in mice both genetically and through metabolite supplementation, and evaluated metabolism, memory, and survival. GlcNAc supplementation in the drinking water had no adverse effect on weight gain in males but increased weight in young female mice. Glucose or insulin tolerance were not affected up to 20 months of age. Of note, we observed improved memory in the Morris water maze in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc supplementation. To assess the effects of genetic HBP activation we overexpressed the key enzyme GFAT1 as well as a constitutively activated point mutant form in all mouse tissues. We detected elevated UDP-GlcNAc levels in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.


title: Sustained upregulation of widespread hippocampal-neocortical coupling following memory encoding

abstract:
Systems consolidation of new experiences into lasting episodic memories involves interactions between hippocampus and the neocortex. Evidence of this process is seen already during early awake post-encoding rest periods. Functional MRI (fMRI) studies have demonstrated increased hippocampal coupling with task-relevant perceptual regions and reactivation of stimulus-specific encoding patterns following intensive encoding tasks. Here we investigate the spatial and temporal characteristics of these hippocampally anchored post-encoding neocortical modulations. Eighty-nine adults participated in an experiment consisting of interleaved memory task- and resting-state periods. As expected, we observed increased post-encoding functional connectivity between hippocampus and individually localized neocortical regions responsive to stimulus categories encountered during memory encoding. Post-encoding modulations were however not restricted to stimulus-selective cortex, but manifested as a nearly system-wide upregulation in hippocampal coupling with all major functional networks. The spatial configuration of these extensive modulations resembled hippocampal-neocortical interaction patterns estimated from active encoding operations, suggesting hippocampal post-encoding involvement by far exceeds reactivation of perceptual aspects. This reinstatement of encoding patterns during immediate post-encoding rest was not observed in resting-state scans collected 12 hours later, nor in control analyses estimating post-encoding neocortical modulations in functional connectivity using other candidate seed regions. The broad similarity in hippocampal functional coupling between online memory encoding and offline post-encoding rest suggests reactivation in humans may involve a spectrum of cognitive processes engaged during experience of an event.

Significance statementStabilization of newly acquired information into lasting memories occurs through systems consolidation - a process which gradually spreads the locus of memory traces from hippocampus to more distributed neocortical representations. One of the earliest signs of consolidation is the upregulation of hippocampal-neocortical interactions during periods of awake rest following an active encoding task. We here show that these modulations involve much larger parts of the brain than previously reported in humans. Comparing changes in hippocampal coupling during post-encoding rest with those observed under active encoding, we find evidence for encoding-like hippocampal reinstatement throughout cortex during task-free periods. This suggests early systems consolidation of an experience involves reactivating not only core sensory details but multiple additional aspects of the encoding event.


title: Globally invariant metabolism but density-diversity mismatch in springtails

abstract: Soil life supports the functioning and biodiversity of terrestrial ecosystems1, 2. Springtails (Collembola) are among the most abundant soil animals regulating soil fertility and flow of energy through above- and belowground food webs3-5. However, the global distribution of springtail diversity and density, and how these relate to energy fluxes remains unknown. Here, using a global dataset collected from 2,470 sites, we estimate total soil springtail biomass at 29 Mt carbon (threefold higher than wild terrestrial vertebrates6) and record peak densities up to 2 million individuals per m{superscript 2} in the Arctic. Despite a 20-fold biomass difference between tundra and the tropics, springtail energy use (community metabolism) remains similar across the latitudinal gradient, owing to the increase in temperature. Neither springtail density nor community metabolism were predicted by local species richness, which was highest in the tropics, but comparably high in some temperate forests and even tundra. Changes in springtail activity may emerge from latitudinal gradients in temperature, predation7, 8, and resource limitation7, 9, 10 in soil communities. Contrasting temperature responses of biomass, diversity and activity of springtail communities suggest that climate warming will alter fundamental soil biodiversity metrics in different directions, potentially restructuring terrestrial food webs and affecting major soil functions.


title: Ecology, more than antibiotics consumption, is the major predictor for the global distribution of aminoglycoside-modifying enzymes

abstract: Antibiotics consumption and its abuses have been historically and repeatedly pointed out as the major driver of antibiotic resistance emergence and propagation. However, several examples show that resistance may persist despite substantial reductions in antibiotic use, and that other factors are at stake. Here we study the temporal, spatial, and ecological distribution patterns of aminoglycoside resistance, by screening more than 160,000 publicly available genomes for 27 clusters of genes encoding aminoglycoside-modifying enzymes (AMEGs). We find that AMEGs are ubiquitous: about 25% of sequenced bacteria carry AMEGs. These bacteria were sequenced from all the continents and terrestrial biomes, and belong to a wide number of phyla. By focusing on several European countries between 1997 and 2018, we show that aminoglycoside consumption has little impact on the prevalence of AMEG-carrying bacteria, whereas most variation in prevalence is observed among biomes. We further analyze the resemblance of resistome compositions across biomes: soil, wildlife, and human samples appear to be central to understand the exchanges of AMEGs between different ecological contexts. Moreover, the genomic distribution of AMEGs suggests a selection for widening resistance spectra, mostly driven by mobile genetic elements. Together, these results support the idea that interventional strategies based on reducing antibiotic use should be complemented by a stronger control of exchanges, especially between ecosystems.

category: genomics


title: Integrating Cell Morphology with Gene Expression and Chemical Structure to Aid Mitochondrial Toxicity Detection

abstract:
Mitochondrial toxicity is an important safety endpoint in drug discovery. Models based solely on chemical structure for predicting mitochondrial toxicity are currently limited in accuracy and applicability domain to the chemical space of the training compounds. In this work, we aimed to utilize both -omics and chemical data to push beyond the state-of-the-art. We combined Cell Painting and Gene Expression data with chemical structural information from Morgan fingerprints for 382 chemical perturbants tested in the Tox21 mitochondrial membrane depolarization assay. We observed that mitochondrial toxicants significantly differ from non-toxic compounds in morphological space and identified compound clusters having similar mechanisms of mitochondrial toxicity, thereby indicating that morphological space provides biological insights related to mechanisms of action of this endpoint. We further showed that models combining Cell Painting, Gene Expression features and Morgan fingerprints improved model performance on an external test set of 236 compounds by 60% (in terms of F1 score) and improved extrapolation to new chemical space. The performance of our combined models was comparable with dedicated in vitro assays for mitochondrial toxicity; and they were able to detect mitochondrial toxicity where Tox21 assays outcomes were inconclusive because of cytotoxicity. Our results suggest that combining chemical descriptors with different levels of biological readouts enhances the detection of mitochondrial toxicants, with practical implications for use in drug discovery.

GRAPHICAL ABSTRACT

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title: Phylogenomics reveals the evolutionary origin of lichenization in chlorophyte algae

abstract:
Mutualistic symbioses, such as lichens formed between fungi and green algae or cyanobacteria, have contributed to major transitions in the evolution of life and are at the center of extant ecosystems. However, our understanding of their evolution and function remains elusive in most cases. Here, we investigated the evolutionary history and the molecular innovations at the origin of lichens in green algae. We de novo sequenced the genomes or transcriptomes of 15 lichen-forming and closely-related non-lichen-forming algae and performed comparative phylogenomics with 22 genomes previously generated. We identified more than 350 functional categories significantly enriched in chlorophyte green algae able to form lichens. Among them, functions such as light perception or resistance to dehydration were shared between lichenizing and other terrestrial algae but lost in non-terrestrial ones, indicating that the ability to live in terrestrial habitats is a prerequisite for lichens to evolve. We detected lichen-specific expansions of glycosyl hydrolase gene families known to remodel cell walls, including the glycosyl hydrolase 8 which was acquired in lichenizing Trebouxiophyceae by horizontal gene transfer from bacteria, concomitantly with the ability to form lichens. Mining genome-wide orthogroups, we found additional evidence supporting at least two independent origins of lichen-forming ability in chlorophyte green algae. We conclude that the lichen-forming ability evolved multiple times in chlorophyte green algae, following a two-step mechanism which involves an ancestral adaptation to terrestrial lifestyle and molecular innovations to modify the partners cell walls.

Significance StatementMutualistic symbioses have contributed to major transitions in the evolution of life and are at the center of extant ecosystems. How these symbiotic associations evolve and function are central questions in biology. Here, we sequenced and compared the genomes of green algal symbionts of the emblematic lichen symbiosis. We discovered functional features specifically expanded in lichen-forming algae suggesting the evolution of a terrestrial lifestyle as a prerequisite for lichen evolution. Projecting the lichen-specific function on the green algae phylogeny support the independent gain of the ability to form lichens in algae, through gene-family expansions and ancient horizontal gene transfer.

category: plant biology


title: basicsynbio and the BASIC SEVA collection: Software and vectors for an established DNA assembly method

abstract: Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore design spaces and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use, while enabling simple hierarchical assemblies through an idempotent format. These features facilitate various applications including pathway engineering, ribosome binding site tuning, fusion protein engineering and multiplexed gRNA expression. In this work we present basicsynbio, an open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio). With basicsynbio, users can access commonly used BASIC parts and linkers while robustly designing new parts and assemblies with exception handling for common design errors. Users can export sequence data and create build instructions for manual or acoustic liquid-handling platforms. The generation of build instructions relies on the BasicBuild Open Standard which is easily parsed for bespoke workflows and is serializable in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio by assembling a collection of 30 vectors using various sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA collection is compatible with BASIC and Golden Gate using BsaI. It encompasses vectors containing six antibiotic resistance markers and five origins of replication from different compatibility groups, including a temperature-sensitive variant. To make the collection accessible we deposited it on Addgene under an OpenMTA agreement. Furthermore, vector sequences are accessible from within the basicsynbio application programming interface along with other collections of parts and linkers, providing an ideal environment to design assemblies for bioengineering applications using BASIC.

category: synthetic biology


title: Multi-micron crisscross structures from combinatorially assembled DNA-origami slats

abstract:
Living systems achieve robust self-assembly across length scales. Meanwhile, nanofabrication strategies such as DNA origami have enabled robust self-assembly of submicron-scale shapes.However, erroneous and missing linkages restrict the number of unique origami that can be practically combined into a single supershape. We introduce crisscross polymerization of DNA-origami slats for strictly seed-dependent growth of custom multi-micron shapes with user-defined nanoscale surface patterning. Using a library of ~2000 strands that can be combinatorially assembled to yield any of ~1e48 distinct DNA origami slats, we realize five-gigadalton structures composed of >1000 uniquely addressable slats, and periodic structures incorporating >10,000 slats. Thus crisscross growth provides a generalizable route for prototyping and scalable production of devices integrating thousands of unique components that each are sophisticated and molecularly precise.

One-sentence summaryCrisscross polymerization of DNA-origami slats can yield micron-scale structures with uniquely addressable nanoscale features.


title: Relapse-like behavior and nAChR sensitization following intermittent access nicotine self-administration

abstract: ABSTRACTMany tobacco smokers consume nicotine intermittently, but the underlying mechanisms and neurobiological changes associated with intermittent nicotine intake are unclear. Understanding intermittent nicotine intake is a high priority, as it could promote therapeutic strategies to attenuate tobacco consumption. We examined nicotine intake behavior and neurobiological changes in male rats that were trained to self-administer nicotine during brief (5 min) trials interspersed with longer (15 min) drug-free periods. Rats readily adapted to intermittent access (IntA) SA following acquisition on a continuous access (ContA) schedule. Probabilistic analysis of IntA nicotine SA suggested reduced nicotine loading behavior compared to ContA, and nicotine pharmacokinetic modeling revealed that rats taking nicotine intermittently may have increased intake to maintain blood levels of nicotine that are comparable to ContA SA. After IntA nicotine SA, rats exhibited an increase in unreinforced responses for nicotine-associated cues (incubation of craving) and specific alterations in the striatal proteome after 7 days without nicotine. IntA nicotine SA also induced nAChR functional upregulation in the interpeduncular nucleus (IPN), and it enhanced nicotine binding in the brain as determined via [11C]nicotine positron emission tomography. Reducing the saliency of the cue conditions during the 5 min access periods attenuated nicotine intake, but incubation of craving was preserved. Together, these results indicate that IntA conditions promote nicotine SA and nicotine seeking after a nicotine-free period.


title: Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues

abstract:
SARS-CoV-2 primarily replicates in mucosal sites, and more information is needed about immune responses in infected tissues. We used rhesus macaques to model protective primary immune responses in tissues during mild COVID-19. Viral RNA levels were highest on days 1-2 post-infection and fell precipitously thereafter. 18F-fluorodeoxyglucose (FDG)-avid lung abnormalities and interferon (IFN)-activated myeloid cells in the bronchoalveolar lavage (BAL) were found on days [~]3-4. Virus-specific effector CD8 and CD4 T cells were detectable in the BAL and lung tissue on days [~]7-10, after viral RNA, lung inflammation, and IFN-activated myeloid cells had declined. Notably, SARS-CoV-2-specific T cells were not detectable in the nasal turbinates, salivary glands, and tonsils on day 10 post-infection. Thus, SARS-CoV-2 replication wanes in the lungs prior to T cell responses, and in the nasal and oral mucosa despite the apparent lack of Ag-specific T cells, suggesting that innate immunity efficiently restricts viral replication during mild COVID-19.

ONE SENTENCE SUMMARYSARS-CoV-2 infection leads to mild, focal lung inflammation, and type I IFN activated myeloid cells that mostly resolve prior to the influx of virus-specific effector T cells or antibody responses in rhesus macaques.

category: immunology


title: The salivary, metal-binding peptide histatin-5 buffers extracellular copper availability

abstract: Antimicrobial peptides (AMPs) are key components of diverse host innate immune systems. The family of human salivary AMPs known as histatins bind Zn and Cu. Fluctuations in Zn and Cu availability play significant roles in the host innate immune response (so-called "nutritional immunity"). Thus, we hypothesised that histatins contribute to nutritional immunity by influencing host Zn and/or Cu availability. We posited that histatins limit Zn availability (promote bacterial Zn starvation) and/or raise Cu availability (promote bacterial Cu poisoning). To test this hypothesis, we examined the interactions between histatin-5 (Hst5) and Group A Streptococcus (GAS), which colonises the human oropharynx. Our results showed that Hst5 does not strongly influence Zn availability. Hst5 did not induce expression of Zn-responsive genes in GAS, nor did it suppress growth of mutant strains that are impaired in Zn transport. Biochemical examination of purified peptides confirmed that Hst5 binds Zn only weakly. By contrast, Hst5 bound Cu tightly and it strongly influenced Cu availability. However, Hst5 did not promote Cu toxicity. Instead, Hst5 suppressed expression of Cu-inducible genes, stopped intracellular accumulation of Cu, and rescued growth of a {Delta}copA mutant strain that is impaired in Cu efflux. We thus proposed a new role for salivary histatins as major Cu buffers in saliva that contribute to microbial homeostasis in the oral cavity and oropharynx by reducing the potential negative effects of Cu exposure (e.g. from food) to microbes. Our results raise broad questions regarding the physiological roles of diverse metal-binding AMPs and the management of host metal availability during host-microbe interactions.

category: microbiology


title: The role of metal binding in the function of the human salivary antimicrobial peptide histatin-5


title: Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

abstract: Spindle orientation is often achieved by a complex of Pins/LGN, Mud/NuMa, Gi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, laser ablation, and depletion of an adherens junction protein disrupted Pins planar polarity and spindle orientation. Furthermore, snail depletion, which abrogates ventral furrow forces, disrupted Pins polarization and spindle orientation, suggesting that morphogenetic movements and resulting forces transmitted through the tissue can polarize Pins and orient division. Thus, morphogenetic forces associated with mesoderm invagination result in planar polarized Pins to mediate division orientation at a distant region of the embryo during morphogenesis. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

category: cell biology


title: The native - exotic plant choice in green roof design: using a multicriteria decision framework to foster urban biodiversity

abstract: Green roofs are considered key elements of the urban green infrastructure since they offer several environmental benefits, including habitat provision for arthropods. To achieve these benefits and ensure green roof success, an appropriate plant selection is an important step in the design of these infrastructures, especially where green roof technology is emerging like in South American cities. So far, decisions of using native or exotic plant species in green roofs had never been evaluated taking into account the plant potential to foster beneficial arthropods. By applying an integrative multicriteria decision framework that combined the habitat template hypothesis with the potential of plants to attract floral visitors and natural enemies, we obtained a ranked set of candidate native and exotic plant species. Among the best-ranked candidate species, we further compared the performance of six native and six exotic species in 30 experimental green roofs installed in Cordoba city, Argentina. To evaluate plant success, the occurrence and cover of each species were recorded one year after establishment under two management conditions: regular watering and weeding of spontaneous plants, and no management (15 roofs each). All selected species increased their vegetative cover one year after establishment. More interestingly, native plants had an advantage over exotic plant species as they exhibited a significantly higher occurrence and a slightly higher cover with no management than exotics. Native annuals were able to reseed the following season even in the absence of management, thus highlighting the relative importance of lifespan as a useful plant trait for future studies in green roof design. Given that green roofs are one of the possible solutions to ameliorate the negative effects of urban habitat loss on arthropod diversity, the development of an integrative multicriteria decision framework that takes into account the potential of native and exotic plant species for promoting beneficial arthropods would give a new twist in plant selection processes for green roofs.


title: Integrated Tick Management in the Upper Midwest: Impact of Invasive Vegetation Removal and Host-targeted Acaricides on Ixodes scapularis Infestation and Borrelia burgdorferi Prevalence of Small Mammals

abstract: Integration of tick management strategies has been suggested to overcome ecological variation in tick, host, pathogen, and habitat, yet empirical evidence assessing combined treatment effect on blacklegged ticks, Ixodes scapularis Say, is limited. In this 5-year study (2014-2018) we tested whether combining two methods targeting tick/mammal interactions could reduce juvenile I. scapularis parasitism of two small mammal species, Peromyscus leucopus Rafinesque and Tamias striatus Linnaeus. Infection of small mammals with Borrelia burgdorferi was used to evaluate host exposure to feeding ticks. Using a factorial design, removal of invasive vegetation (Amur honeysuckle, Lonicera maackii Ruprecht and common buckthorn, Rhamnus cathartica Linnaeus) was coupled with deployments of permethrin-treated cotton nesting materials (tick tubes) and evaluated against control sites. Removal of invasive vegetation resulted in lower captures of T. striatus suggesting that treatment impacted reservoir activity in the plots. Deployments of permethrin-treated cotton were effective at reducing the frequency of juvenile I. scapularis parasitism of P. leucopus by 91% across the study compared to controls. However, tick tubes did not offer consistent protection against mouse exposure to B. burgdorferi exposure. An additive negative effect was detected for juvenile tick intensity on P. leucopus when tick tubes were combined with invasive vegetation removal. We conclude that integration of these two methods provides very limited benefit and that permethrin treatment alone offers the best option for reducing I. scapularis infestation on P. leucopus.


title: On the environmental zero-point problem in microevolutionary climate response predictions

abstract: It is well documented that individuals of wild populations can adjust to climate change by means of phenotypic plasticity, but few reports on adaptation by means of genetically based microevolution caused by selection. Disentanglement of these separate effects requires that the reference environment (the environmental zero-point) is defined, and this should not be done arbitrarily. The problem is that an error in the reference environment may lead to large errors in predicted microevolution. Together with parameter values and initial mean trait values, the reference environment can be estimated from environmental, phenotypic and fitness data. A prediction error method for this purpose is described, with the feasibility shown by simulations. As shown in a toy example, an estimated reference environment may have large errors, especially for small populations. This may still be a better choice than use of an initial environmental value in a recorded time series, or the mean value, which is often used. Another alternative may be to use the mean value of a past and stationary stochastic environment, which the population is judged to have been fully adapted to, in the sense that the expected geometric mean fitness was at a global maximum. Exceptions are cases with constant phenotypic plasticity, where the microevolutionary changes per generation follow directly from phenotypic and environmental data, independent of the chosen reference environment.

category: evolutionary biology


title: The important choice of reference environment in microevolutionary climate response predictions


title: Spread of new mutations through space

abstract: The terms population size and population density are often used interchangeably, when in fact they are quite different. When viewed in a spatial landscape, density is defined as the number of individuals within a square unit of distance, while population size is simply the total count of a population. In discrete population genetics models, the effective population size is known to influence the interaction between selection and random drift with selection playing a larger role in large populations while random drift has more influence in smaller populations. Using a spatially explicit simulation software we investigate how population density affects the flow of new mutations through a geographical space. Using population density, selectional advantage, and dispersal distributions, a model is developed to predict the speed at which the new allele will travel, obtaining more accurate results than current diffusion approximations provide. We note that the rate at which a neutral mutation spreads begins to decay over time while the rate of spread of an advantageous allele remains constant. We also show that new advantageous mutations spread faster in dense populations.


title: Covariant Fitness Clusters Reveal Structural Evolution of SARS-CoV-2 Polymerase Across the Human Population

abstract: Understanding the fitness landscape of viral mutations is crucial for uncovering the evolutionary mechanisms contributing to pandemic behavior. Here, we apply a Gaussian process regression (GPR) based machine learning approach that generates spatial covariance (SCV) relationships to construct stability fitness landscapes for the RNA-dependent RNA polymerase (RdRp) of SARS- CoV-2. GPR generated fitness scores capture on a residue-by-residue basis a covariant fitness cluster centered at the C487-H642-C645-C646 Zn2+ binding motif that iteratively evolves since the early phase pandemic. In the Alpha and Delta variant of concern (VOC), multi-residue SCV interactions in the NiRAN domain form a second fitness cluster contributing to spread. Strikingly, a novel third fitness cluster harboring a Delta VOC basal mutation G671S augments RdRp structural plasticity to potentially promote rapid spread through viral load. GPR principled SCV provides a generalizable tool to mechanistically understand evolution of viral genomes at atomic resolution contributing to fitness at the pathogen-host interface.


title: Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers

abstract:
BackgroundThe transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4{degrees}C would cause RNA degradation resulting in altered transcriptome profiles.

ResultsWe produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after one, three, six, or eight hours. However, samples processed after 24 hours of storage had a lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3/5 bias revealed no differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24-hour and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour.

ConclusionsThe PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.


title: Improving AlphaFold modeling using implicit information from experimental density maps

abstract: Machine learning prediction algorithms such as AlphaFold1 and RoseTTAFold2 can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy3-6. We hypothesized that by implicitly including experimental information, a greater portion of a model could be predicted accurately, and that this might synergistically improve parts of the model that were not fully addressed by either machine learning or experiment alone. An iterative procedure was developed in which AlphaFold models are automatically rebuilt based on experimental density maps and the rebuilt models are used as templates in new AlphaFold predictions. We find that including experimental information improves prediction beyond the improvement obtained with simple rebuilding guided by the experimental data. This procedure for AlphaFold modeling with density has been incorporated into an automated procedure for crystallographic and electron cryo-microscopy map interpretation.

category: biochemistry


title: Improved AlphaFold modeling with implicit experimental information


title: Climate Surveys of Biomedical PhD Students and Training Faculty Members in the Time of Covid

abstract: In July 2020, four months into the disruption of normal life caused by the Covid-19 pandemic, we assessed the institutional climate within the School of Medicine. Voluntary surveys were completed by 135 graduate students in 11 PhD-granting programs and by 83 members of the graduate training faculty. Several themes emerged. PhD students work hard, but the number of hours spent on research-related activities has declined during the pandemic. The students are worried about the pandemics impact on their research productivity, consequent delays in their graduation, and diminished future job prospects. Many late stage PhD students feel they do not have adequate time or resources to plan for their future careers. Symptoms of anxiety and/or depression are prevalent in 51% of the students, based on answers to standardized questions. Most students report they have strong mentoring relationships with their faculty advisors and like their programs, but they identify to a lesser extent with the medical school as a whole. Faculty think highly of their graduate students and are also worried about the pandemics impact upon productivity and the welfare of students. Students are interested in access to an Ombuds office, which is currently being organized by the medical school. Moving forward, the school needs to address issues of bias, faculty diversity, support for mentor training, professional development, and the imposter syndrome. We must also work to create a climate in which many more graduate students feel that they are valued members of the academic medicine community.

category: scientific communication and education


title: Homologous recombination between tandem paralogues drives evolution of Type VII secretion system immunity genes in firmicute bacteria

abstract: The Type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified in Staphylococcus aureus, EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identical esaG and tsaI immunity genes are encoded in all S. aureus strains. During genome sequence analysis of closely related S. aureus strains we noted that there had been a deletion of six consecutive esaG copies in one lineage. To investigate this further, we analysed the sequences of the tandem esaG genes and their encoded proteins. We identified three blocks of high sequence homology shared by all esaG genes, and identified evidence of extensive recombination events between esaG paralogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss of esaG genes from S. aureus genomes. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence homology locate to the 5 and 3 end of tipC genes, and we found strong evidence for recombination between tipC paralogues encoded by Streptococcus mitis BCC08. By contrast, we found only a single block of homology across tsaI genes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.


title: Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

abstract:
Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an [~]171 bp -satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto -satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the -satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the -satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces.

One-Sentence SummaryThe human inner kinetochore CCAN complex tightly grips the linker DNA of the -satellite CENP-A nucleosome.


title: Frequency Effects on Spelling Error Recognition: An ERP Study

abstract: Spelling errors are ubiquitous in all writing systems. Most studies exploring spelling errors focused on the phonological plausibility of errors. However, unlike typical pseudohomophones, spelling errors occur in naturally produced written language with variable frequencies. We investigated the time course of recognition of the most frequent orthographic errors in Russian (error in an unstressed vowel at the root) and the effect of word frequency on this process. During ERP recording, 26 native Russian speakers silently read high-frequency correctly spelled words, low-frequency correctly spelled words, high-frequency words with errors, and low-frequency words with errors. The amplitude of P200 was more positive for correctly spelled words than for misspelled words and did not depend on the frequency of the words. Word frequency affected spelling recognition in the later stages of word processing (350-700 ms): high-frequency misspelled words elicited a greater P300 than high-frequency correctly spelled words, and low-frequency misspelled words elicited a greater N400 than low-frequency correctly spelled words. We observe spelling effects in the same time window for both the P300 and N400, which may reflect temporal overlap between mainly categorization processes based on orthographic properties for high-frequency words and phonological processes for low-frequency words. We concluded that two independent pathways can be active simultaneously during spelling recognition: one reflects mainly orthographic processing of high-frequency words and the other is the phonological processing of low-frequency words. Our findings suggest that these pathways are associated with different ERP components. Therefore, our results complement existing reading models and demonstrate that the neuronal underpinnings of spelling error recognition during reading depend on word frequency.


title: Time-adaptive Unsupervised Auditory Attention Decoding Using EEG-based Stimulus Reconstruction

abstract: The goal of auditory attention decoding (AAD) is to determine to which speaker out of multiple competing speakers a listener is attending based on the brain signals recorded via, e.g., electroencephalography (EEG). AAD algorithms are a fundamental building block of so-called neuro-steered hearing devices that would allow identifying the speaker that should be amplified based on the brain activity. A common approach is to train a subject-specific stimulus decoder that reconstructs the amplitude envelope of the attended speech signal. However, training this decoder requires a dedicated  ground-truth EEG recording of the subject under test, during which the attended speaker is known. Furthermore, this decoder remains fixed during operation and can thus not adapt to changing conditions and situations. Therefore, we propose an online time-adaptive unsupervised stimulus reconstruction method that continuously and automatically adapts over time when new EEG and audio data are streaming in. The adaptive decoder does not require ground-truth attention labels obtained from a training session with the end-user and instead can be initialized with a generic subject-independent decoder or even completely random values. We propose two different implementations: a sliding window and recursive implementation, which we extensively validate on three independent datasets based on multiple performance metrics. We show that the proposed time-adaptive unsupervised decoder outperforms a time-invariant supervised decoder, representing an important step toward practically applicable AAD algorithms for neuro-steered hearing devices.


title: Comprehensive analysis of disease pathology in immunocompetent and immunocompromised hamster models of SARS-CoV-2 infection

abstract: The pathogenesis of SARS-CoV-2 in the context of a specific immunological niche is not fully understood. Here, we used a golden Syrian hamster model to systematically evaluate the kinetics of host response to SARS-CoV-2 infection, following disease pathology, viral loads, antibody responses, and inflammatory cytokine expression in multiple organs. The kinetics of SARS-CoV-2 pathogenesis and genomewide lung transcriptome was also compared between immunocompetent and immunocompromised hamsters. We observed that the body weight loss was proportional to the SARS-CoV-2 infectious dose and lasted for a short time only in immunocompetent hamsters. Body weight loss was more prominent and prolonged in infected immunocompromised hamsters. While the kinetics of viral replication and peak live viral loads were not significantly different at low and high infectious doses (LD and HD), the HD-infected immunocompetent animals developed severe lung disease pathology. The immunocompetent animals cleared the live virus in all tested tissues by 12 days post-infection and generated a robust serum antibody response. In contrast, immunocompromised hamsters mounted an inadequate SARS-CoV-2 neutralizing antibody response, and the virus was detected in the pulmonary and multiple extrapulmonary organs until 16 days post-infection. These hamsters also had prolonged moderate inflammation with severe bronchiolar-alveolar hyperplasia/metaplasia. Consistent with the difference in disease presentation, distinct changes in the expression of inflammation and immune cell response pathways and network genes were seen in the lungs of infected immunocompetent and immunocompromised animals. This study highlights the interplay between the kinetics of viral replication and the dynamics of SARS-CoV-2 pathogenesis at organ-level niches and maps how COVID-19 symptoms vary in different immune contexts. Together, our data suggest that the histopathological manifestations caused by progressive SARS-CoV-2 infection may be a better predictor of COVID-19 severity than individual measures of viral load, antibody response, and cytokine storm at the systemic or local (lungs) levels in the immunocompetent and immunocompromised hosts.

category: pathology


title: A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

abstract: Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.


title: Ecological speciation promoted by divergent regulation of functional genes within African cichlid fishes

abstract: Rapid ecological speciation along depth gradients has taken place independently and repeatedly in freshwater fishes. While the extent of genomic divergence between ecomorphs is often well understood, the molecular mechanisms facilitating such rapid diversification are typically unclear. In Lake Masoko, an East African crater lake, the cichlid Astatotilapia calliptera has diverged into shallow littoral and deep benthic ecomorphs with strikingly different jaw structures within the last 1,000 years. Using genome-wide transcriptome data from jaw tissue, we explore two major regulatory transcriptional mechanisms, expression and splicing QTL variants and examine their contribution to differential gene expression underpinning functional phenotypes. We identified 7,550 genes with significant differential expression between ecomorphs, of which 4.2% were regulated by cis-regulatory expression QTLs, and 6.4% were regulated by cis-regulatory splicing QTLs. There were also strong signals of divergent selection of differentially expressed genes that showed divergent regulation from expression, splicing or both QTL variants, including genes associated with major jaw plasticity and adaptation networks, adaptive immune system response, and oxidoreductase processes. These results suggest that transcriptome plasticity and modification have important roles during early-stage ecological speciation and demonstrate the role of regulatory-variants as important targets of selection driving ecologically-relevant divergence in gene expression that is associated with adaptive diversification.


title: Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR

abstract: Ligand binding to the EGF receptor (EGFR) triggers multiple signal transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its crosstalk with signaling are poorly understood. Here, we combined peroxidase-catalyzed proximity labeling, isobaric peptide tagging and quantitative mass-spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identified a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We showed that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, was enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes, and demonstrated that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.

category: systems biology


title: Digenic traits under population admixture and inbreeding

abstract: The population genetics of digenic genotypes in diploid populations, genotypes based on alleles at two loci, have been studied theoretically for decades with relevant digenic traits of medical interest being known for over 25 years. Given the effects of linkage and linkage disequilibrium on two locus genotypes, it should be expected that these factors can change the expected frequencies of digenic genotypes in many, sometimes unexpected, ways. In particular, the combination of linkage disequilibrium and inbreeding can combine to increase the frequencies of double homozygotes and double heterozygotes significantly over outbred comparisons. Given the prevalence of linkage disequilibrium in recently admixed populations, this can lead to large shifts in trait prevalence such that it can sometimes exceed that of either original pre-admixed population with the combined effects of linkage disequilibrium and inbreeding. Here we investigate the frequencies of digenic genotypes under the combined effects of linkage, linkage disequilibrium, and inbreeding to analyze how these interact to increase or decrease the frequency of the genotypes across two loci.


title: Regulation of Chromatin Accessibility by hypoxia and HIF

abstract: Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) coordinate a transcriptional programme required for survival and adaptation to hypoxia. The effects of hypoxia and HIF on the chromatin accessibility landscape are still unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility enriched at hypoxia transcriptionally responsive genes. These changes are predominantly HIF dependent, reversible upon reoxygenation and partially mimicked by chemical HIF stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that indeed, HIF stabilisation is necessary and sufficient to alter chromatin accessibility in hypoxia, with implications for our understanding of gene expression regulation by hypoxia and HIF.

category: molecular biology


title: Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus

abstract: RNA interference (RNAi) is a conserved mechanism of small RNA-mediated genome regulation commonly involved in suppression of transposable elements (TEs) through both post-transcriptional silencing, and transcriptional repression via heterochromatin assembly. The fission yeast Schizosaccharomyces pombe has been extensively utilised as a model for studying RNAi pathways. However, this species is somewhat atypical in that TEs are not major targets of RNAi, and instead small RNAs correspond primarily to non-coding pericentromeric repeat sequences, reflecting a specialised role for the pathway in promoting heterochromatin assembly in these regions. In contrast, in the related fission yeast Schizosaccharomyces japonicus, sequenced small RNAs correspond primarily to TEs. This suggests there may be fundamental differences in the operation of RNAi pathways in these two related species. To investigate these differences, we probed RNAi function in S. japonicus. Unexpectedly, and in contrast to S. pombe, we found that RNAi is essential in this species. Moreover, viability of RNAi mutants can be rescued by mutations implicated in enhanching RNAi-independent heterochromatin propagation. These rescued strains retain heterochromatic marks on TE sequences, but exhibit derepression of TEs at the post-transcriptional level. Our findings indicate that S. japonicus retains the ancestral role of RNAi in facilitating suppression of TEs via both post-transcriptional silencing and heterochromatin assembly, with specifically the heterochromatin pathway being essential for viability, likely due to a function in genome maintenance. The specialised role of RNAi in heterochromatin assembly in S. pombe appears to be a derived state that emerged after the divergence of S. japonicus.


title: Signalling centre vortices coordinate collective behaviour in social amoebae

abstract: Self-sustaining signalling waves provide a source of information in living systems. A classic example is the rotating spiral waves of cAMP (chemoattractant) release that encode Dictyostelium morphogenesis. These patterns remain poorly characterised due to limitations in tracking the signalling behaviour of individual cells in the context of the whole collective. Here, we have imaged Dictyostelium populations over millimetre length scales and track the emergence, structure, progression and biological effects of cAMP waves by monitoring the signalling states and motion of individual cells. Collective migration coincides with a decrease in the period and speed of waves that stem from an increase in the rotational speed and curvature of spiral waves. The dynamics and structure of spiral waves are generated by the vortex motion of the spiral tip. Spiral tip circulation spatially organises a small group of cells into a ring pattern, which also constrains spiral tip motion. Both the cellular ring and tip path gradually contract over time, resulting in the acceleration of spiral rotation and change in global wave dynamics. Aided by mathematical modelling, we show that this contraction is due to an instability driven by a deflection in cell chemotaxis around the spiral tip cAMP field, resulting in a deformation of the cellular ring pattern towards its centre. That is, vortex contraction modulates the source of information which, upon dissemination (excitable signal relay) and decoding (chemotaxis), triggers morphogenesis. By characterising rotating spiral waves at this level of detail, our results describe a mechanism by which information generated by a self-sustaining signal, and disseminated across the population, is modulated at the organisational source.

category: biophysics


title: Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

abstract:
The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, 1,3-fucosyltransferase and fucosyl 4- O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface- exposed lipid virulence factors.

IMPORTANCEM. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogens ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacteria, but its existence is only known in a non- pathogenic model, M. smegmatis. Here, we demonstrated the presence of the IMD in M. tuberculosis, making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD is the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.


title: Dynamic changes in mRNA nucleocytoplasmic localization in the nitrate response of Arabidopsis roots

abstract: Nitrate (NO3-) is a signaling molecule that regulates gene expression in plants. The nitrate response has been extensively characterized at the transcriptome level. However, we know little about RNA nucleocytoplasmic dynamics during nitrate response. To understand the role of mRNA localization during the nitrate response, we isolated mRNA from the nucleus, cytoplasm, and whole-cells from nitrate-treated Arabidopsis roots and performed RNA-seq. We identified 402 differentially localized transcripts (DLTs) in response to nitrate. DLTs were enriched in GO-terms related to metabolism, response to stimulus, and transport. DLTs showed five localization patterns: nuclear reduction, cytoplasmic reduction, nuclear accumulation, cytoplasmic accumulation, or delayed-cytoplasmic accumulation in response to nitrate. DLTs exhibited large changes in RNA polymerase II occupancy of cognate genes and high mRNA turnover rates, indicating these are rapidly replaced mRNAs. The NITRATE REDUCTASE 1 (NIA1) transcript exhibited the largest changes in synthesis and decay. Using single-molecule RNA FISH, we showed that NIA1 nuclear accumulation occurs mainly at transcription sites. The decay profiles for NIA1 showed a higher half-life when the transcript accumulated in the nucleus than in the cytoplasm. We propose that regulating nucleocytoplasmic mRNA distribution allows tuning transcript availability of fastly replaced mRNAs, controlling plants adaptive response to nitrogen nutrient signals.


title: Wnt11 family dependent morphogenesis during frog gastrulation is marked by the cleavage furrow protein anillin

abstract: Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down zygotic Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes at the tailbud stage. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues - including the archenteron - by imaging intact embryos stained for anillin and microtubules. The cleavage furrow protein anillin provided an exceptional cytological marker for blastopore lip and archenteron morphogenesis and the consequent disruption through loss of Wnt 11 signaling. These cytological changes suggest a novel role for the regulation of contractility and stiffness of the epithelial cells that result in dramatic shape changes and are important in gastrulation.

category: developmental biology