title: Salient 40 Hz sounds probe affective aversion and neural excitability

abstract: The human auditory system is not equally reactive to all frequencies of the audible spectrum. Emotional and behavioral reactions to loud or aversive acoustic features can vary from one individual to another, to the point that some exhibit exaggerated or even pathological responses to certain sounds. The neural mechanisms underlying these interindividual differences remain unclear. Whether distinct aversion profiles map onto neural excitability at the individual level needs to be tested. Here, we measured behavioral and EEG responses to click trains (from 10 to 250 Hz, spanning the roughness and pitch perceptual ranges) to test the hypothesis that interindividual variability in aversion to rough sounds is reflected in neural response differences between participants. Linking subjective aversion to 40 Hz steady-state EEG responses, we demonstrate that participants experiencing enhanced aversion to roughness also show stronger neural responses to this attribute. Interestingly, this pattern also correlates with inter-individual anxiety levels, suggesting that this personality trait might interact with subjective sensitivity and neural excitability to these sounds. These results support the idea that 40 Hz sounds can probe the excitability of non-canonical auditory systems involved in exogenous salience processing and aversive responses at the individual level. By linking subjective aversion to neural excitability, 40 Hz sounds provide neuromarkers relevant to a variety of pathological conditions, such as those featuring enhanced emotional sensitivity (hyperacusis, anxiety) or aberrant neural responses at 40 Hz (autism, schizophrenia).


title: The role of floral traits in community assembly process at high elevations in Lesser Himalaya

abstract:
O_LIEcological theory postulates that plant trait research should consider multiple traits related to different organs and/or ontogenetic stages as such traits represent different ecological niche axes. Particularly, floral traits have been suggested to play an important role in assembling plant communities along environmental gradients as they determine the reproductive success, one of the key functions in plants. Yet, the predictive power of floral traits in community assembly research remains largely unverified empirically.
C_LIO_LIWe analyzed the predictive power of six floral traits of 139 herbaceous species for inferring community assembly process in twenty-one sites located along an elevation gradient in Lesser Himalaya ranging from 2,000 to 4,000 meters above sea level. The floral trait variability along the gradient was analyzed using community-weighted trait mean (CWM) values and functional diversities (FD) calculated for each of the study communities.
C_LIO_LIThe CWM values for onset of flowering and flower display area increased significantly with increasing elevation, whereas specific flower area showed an opposite pattern. In combination with convergence in onset of flowering and specific area (i.e., lower FD values in high elevation sites), these patterns suggest that abiotic filtering and plant-pollinator interactions affected the floral trait composition of the communities studied. Increasing low-temperature stress towards high-elevation sites selected for late-flowering species that produce resource-intensive flowers with larger display areas.
C_LIO_LILow pollinator abundancy and activity in high elevation, could also explain why these traits were selected in the study communities. Delayed flowering with increasing elevations might facilitate the phenological overlap of plants and their pollinators, as pollinator activity at higher elevation peaks in the second half of the vegetation period. The dominance of species with low specific flower area and larger display area in high elevation communities were attributed to the increased flower longevity and attraction of pollinators, respectively, to maximize pollination success under pollinator scarcity.
C_LIO_LISynthesis. Our study provides empirical support of the recent argument that floral traits contribute considerably to the assembly of plant communities along environmental gradients. Thus, such traits should be included into community assembly research agenda as they represent key growth and survival ecological functions.
C_LI

category: ecology


title: Converting cropping systems into seasonal habitat functionality reveals the hump-shaped responses of carabid beetles to agricultural management

abstract:
O_LIUnderstanding effects on the huge diversity of cropping systems on local biodiversity is challenging but necessary to implement agroecological systems. Through a functional approach, the translation of cropping systems into resource and disturbance gradients is promising to decipher the relationship between cropping systems and biodiversity but has never been implemented for arthropods.
C_LIO_LITo investigate contributions of resource and disturbance gradients arising from cropping systems vs environmental context (regional effect, meteorological conditions and landscape characteristics) on beneficial arthropod communities, we used a dataset collected in 60 crop fields from three French areas over a five-years period. It includes all farmers interventions, crop sequences, meteorological data, landscape composition and carabid samplings.
C_LIO_LIWe found that the environmental context contributed to about 75% of explained carabid variations on average, while resource and disturbance gradients contributed to about 25% of explained carabid variations. The resource and disturbance gradients were particularly important in winter and spring preceding the spring-summer period to determine carabid variations.
C_LIO_LIMoreover, we identified thresholds above which resource and disturbance gradients start being beneficial or detrimental for carabids. For example, a Treatment Frequency Index above 2.07 in spring decreased the total activity density of carabids during the spring-summer period.
C_LIO_LISynthesis and application. While implementing for the first time a functional approach to understand the effects of different facets of cropping systems on arthropods, our study also allows us to identify periods and thresholds above which specific practices affect carabids. The identification of such thresholds can guide the provision of recommendations for policy, stakeholders and farmers about how to reduce cropping systems impact on arthropods.
C_LI


title: Fusing spheroids to aligned μ-tissues in a Heart-on-Chip featuring oxygen sensing and electrical pacing capabilities

abstract: Over the last decade Organ-on-Chip (OOC) emerged as a promising technology for advanced in vitro models, recapitulating key physiological cues. OOC approaches tailored for cardiac tissue engineering resulted in a variety of platforms, some of which integrate stimulation or probing capabilities. Due to manual handling processes, however, a large-scale standardized and robust tissue generation, applicable in an industrial setting, is still out of reach. Here, we present a novel cell injection and tissue generation concept relying on spheroids, which can be produced in large quantities and uniform size from induced pluripotent stem cell-derived human cardiomyocytes. Hydrostatic flow transports and accumulates spheroids in dogbone-shaped cultivation chambers, which subsequently fuse and form aligned, contracting cardiac muscle fibers. Furthermore, we demonstrate electrical stimulation capabilities by utilizing fluidic media connectors as electrodes and provide the blueprint of a low-cost, open-source, scriptable pulse generator. We report on a novel integration strategy of optical O2 sensor spots into resin-based microfluidic systems, enabling in situ determination of O2 partial pressures. Finally, proof-of-concept demonstrating electrical stimulation combined with in situ monitoring of metabolic activity in cardiac tissues is provided. The developed system thus opens the door for advanced OOCs integrating biophysical stimulation as well as probing capabilities and serves as blueprint for the facile and robust generation of high density microtissues in microfluidic modules amenable for scale-up and automation.

category: bioengineering


title: Clinically oriented prediction of patient response to targeted and immunotherapies from the tumor transcriptome

abstract: Precision oncology is gradually advancing into mainstream clinical practice, demonstrating significant survival benefits. However, eligibility and response rates remain limited in many cases, calling for better predictive biomarkers. Here we present ENLIGHT, a transcriptomics-based computational approach that identifies clinically relevant genomic interactions and uses them to predict patients response to a variety of therapies in multiple cancer types without training on previous response data. We study ENLIGHT in two translationally oriented scenarios, Personalized Medicine (PM), aimed at prioritizing treatments for a single patient, and Clinical Trial Design (CTD), selecting the most likely responders in a patient cohort. Evaluating ENLIGHTs PM performance on 21 blinded clinical trial datasets, we show that it can effectively predict treatment response across multiple therapies and cancer types (obtaining an odds ratio of 2.59), substantially improving upon SELECT, a previously published transcriptomics-based approach, and performing as well as supervised predictors developed for specific indications and drugs, but on a much broader array of therapies and indications. In the CTD scenario, ENLIGHT can markedly enhance the success of clinical trials by excluding non-responders while achieving more than 90% of the response rate attainable under an optimal exclusion strategy. In sum, ENLIGHT is one of the first approaches to demonstrably predict therapeutic response across multiple cancer types by leveraging the transcriptome.

category: bioinformatics


title: UPP2: Fast and Accurate Alignment Estimation of Datasets with Fragmentary Sequences

abstract:
MotivationMultiple sequence alignment (MSA) is a basic step in many bioinformatics pipelines. However, achieving highly accurate alignments on large datasets, especially those with sequence length heterogeneity, is a challenging task. UPP (Ultra-large multiple sequence alignment using Phylogeny-aware Profiles) is a method for MSA estimation that builds an ensemble of Hidden Markov Models (eHMM) to represent an estimated alignment on the full length sequences in the input, and then adds the remaining sequences into the alignment using selected HMMs in the ensemble. Although UPP provides good accuracy, it is computationally intensive on large datasets.

ResultsWe present UPP2, a direct improvement on UPP. The main advance is a fast technique for selecting HMMs in the ensemble that allows us to achieve the same accuracy as UPP but with greatly reduced runtime. We show UPP2 produces more accurate alignments compared to leading MSA methods on datasets exhibiting substantial sequence length heterogeneity, and is among the most accurate otherwise.

Availabilityhttps://github.com/gillichu/sepp

Contactwarnow@illinois.edu


title: Tween-20 induces the structural remodelling of single lipid vesicles

abstract: The interaction of Tween-20 with lipid membranes is crucial for a number of biotechnological applications including viral inactivation and membrane protein extraction, but the underlying mechanistic details have remained elusive. Evidence from ensemble assays supports a global model of Tween-20 induced membrane disruption that broadly encompasses association of the surfactant with the membrane surface, membrane fragmentation and the release of mixed micelles to solution, but whether this process involves intermediate and dynamic transitions between regimes is an open question. In search of the mechanistic origins of membrane disruption, increasing focus is put on identifying Tween-20 interactions with highly controllable model membranes. In light of this, and to unveil quantitative mechanistic details, we employed highly interdisciplinary biophysical approaches, including quartz-crystal microbalance with dissipation monitoring, steady-state and time-resolved fluorescence and FRET spectroscopy, dynamic light scattering, fluorescence correlation spectroscopy, wide-field single-vesicle imaging and scanning electron microscopy, to interrogate the interactions between Tween-20 and both freely-diffusing and surface-immobilized model-membrane vesicles. Using ultrasensitive sensing approaches, we discovered that Tween-20 leads to a stepwise and phase-dependent structural remodelling of sub-micron sized vesicles that includes permeabilization and swelling, even at detergent concentrations below the critical micellar concentration. These insights into the structural perturbation of lipid vesicles upon Tween-20 interaction highlight the impact on vesicle conformation prior to complete solubilization, and the tools presented may have general relevance for probing the interaction between lipid vesicles and a wide variety of disruptive agents.

category: biophysics


title: Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes

abstract: Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.


title: An Open-source, Cost-efficient Excitation Module for Single-molecule Microscopy

abstract: In fluorescence microscopy, scientific-grade laser diodes are key components and contribute a considerable expense to the total cost of the microscope setup. Existing open-source laser excitation modules are mainly designed for super-resolution imaging and have not been fully characterised to meet the requirements of single-molecule spectroscopy measurements. In this paper we introduce an open-source, cost-efficient excitation module that can be used for confocal, accurate single-molecule FRET, fluorescence correlation spectroscopy (FCS) and low-power super-resolution techniques. The module delivers two diode lasers (520 nm and 638 nm) via a single-mode fibre, with stable power output (<1% deviation), which can be modulated on the microsecond timescale. Here, we benchmark these lasers against smFRET standards, and recover consistent diffusion coefficients from FCS measurements, thereby demonstrating their suitability for a range of single-molecule spectroscopic experiments, whilst reducing the overall costs of our open-source smFRET instrument (the smfBox) by ~50%, making these types of experiments even more accessible to the widest possible userbase.


title: Blood-brain barrier permeability to enrofloxacin in Pengze crucian carp (Carassius auratus var. Pengze)

abstract: To explore the permeability of blood-brain barrier (BBB) to enrofloxacin (ENR) and the role of brain injury markers in the evaluation of brain damage, crucian carp (Carassius auratus var. Pengze) was employed in this work as the research object treated by a single oral administration of the antibiotic. Results showed that ENR residues could be detected in brain of crucian carp exposed to both half lethal dose (LD50) and safe dose (SD) of ENR throughout the trial period, indicating that ENR had an ability of permeating the BBB of crucian carp. Evans blue (EB) dispersively distributed in brain tissue of fish exposed to SD ENR, but not in the vehicle-treated fish brain, which suggested structural failure of BBB by ENR. Expression levels of BBB-related molecules, occludin and P-glycoprotein (P-gp), were significantly down-regulated by ENR. Brain injury markers, NSE, S100B and GFAP were improved that were demonstrated by elevated contents of proteins in brain and serum. According to the data of correlation analysis, it was observed that ENR residue was closely related to S100B and GFAP, indicating that S100B and GFAP could be used as evaluation indicators for brain injury of crucian carp. All findings mentioned above suggest that ENR may cause structural change of BBB, resulting in biological brain damage in crucian carp.

category: pharmacology and toxicology


title: Hidden potential of the supporting scaffold as a structural module for plant cystatin design

abstract: Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins towards herbivorous pest digestive Cys proteases, typically involving sequence alterations in the inhibitory loops and/or N-terminal trunk of the protein interacting with specific amino acid residues of the target protease. In this study, we assessed whether the loops-supporting frame, or scaffold, would represent a valuable structural module for cystatin function improvement. Twenty hybrid cystatins were designed in silico, consisting of the N-terminal trunk and two inhibitory loops of a given donor cystatin grafted onto the scaffold of an alternative, recipient cystatin. Synthetic genes for the hybrids were expressed in E. coli, and the resulting proteins assessed for their potency to inhibit model Cys protease papain and the digestive Cys proteases of Colorado potato beetle (Leptinotarsa decemlineata) used as an insect pest model. In line with the occurrence of positively selected amino acids presumably influencing inhibitory activity in the scaffold region of plant cystatins, grafting the N-terminal trunk and inhibitory loops of a given cystatin onto the scaffold of an alternative cystatin generally had an effect on the inhibitory potency of these function-related elements against Cys proteases. For instance, hybrid cystatins including the three structural elements of model tomato cystatin SlCYS8 grafted on the scaffold of cystatins from other plant families showed Ki values altered by up to 3-fold for papain, and inhibitory efficiencies increased by up to 8-fold against L. decemlineata cathepsin L-like proteases, compared to wild-type SlCYS8 bearing the original scaffold. Our data point to a significant influence of the cystatin scaffold on the inhibitory activity of the N-terminal trunk and protease inhibitory loops. They also suggest the potential of this structural element as a module for plant cystatin design to generate functional variability against Cys proteases, including the digestive proteases of herbivorous pests.

category: plant biology


title: Regulatory strategies to schedule threshold crossing of protein levels at a prescribed time

abstract: The timing of diverse cellular processes is based on the instant when the concentration of regulatory proteins crosses a critical threshold level. Hence, noise mechanisms inherent to these protein synthesis pathways drive statistical fluctuations in such events timing. How to express proteins ensuring both the threshold crossing at a prescribed time and minimal timing fluctuations? To find this optimal strategy, we formulate a model where protein molecules are synthesized in random bursts of gene activity. The burst frequency depends on the protein level creating a feedback loop, and cellular growth dilutes protein concentration between consecutive bursts. Counterintuitively, our analysis shows that positive feedback in protein production is best for minimizing variability in threshold-crossing times. We analytically predict the optimal feedback strength in terms of the dilution rate. As a corollary to our result, a no-feedback strategy emerges as the optimal strategy in the absence of dilution. We further consider other noise sources, such as randomness in either the initial condition or the threshold level, and find that in many cases, we need either strongly negative or positive feedback for precise scheduling for events.

category: systems biology


title: Tissue-specific landscape of protein aggregation and quality control in an aging vertebrate

abstract:
Protein aggregation is a hallmark of age-related neurodegeneration. Yet, aggregation during normal aging and in tissues other than the brain is poorly understood. Here we leverage the African turquoise killifish to systematically profile protein aggregates in seven tissues of an aging vertebrate. Age-dependent aggregation is strikingly tissue-specific, and not simply driven by protein expression differences. Experimental interrogation, combined with machine learning, indicates that this specificity is linked to both protein-autonomous biophysical features and tissue-selective alterations in protein quality control. Co-aggregation of protein quality control machinery during aging may further reduce proteostasis capacity, exacerbating aggregate burden. A segmental progeria model with accelerated aging in specific tissues exhibits selectively increased aggregation in these same tissues. Intriguingly, many age-related protein aggregates arise in wild-type proteins that, when mutated, drive human diseases. Our data chart a comprehensive landscape of protein aggregation during aging and reveal strong, tissue-specific associations with dysfunction and disease.

HIGHLIGHTSO_LITissue-specific protein aggregation is prevalent during vertebrate aging
C_LIO_LIBoth protein biophysical properties and tissue-specific protein homeostasis patterns impact aggregation
C_LIO_LIA segmental progeria model with accelerated aging exhibits selectively increased protein aggregation in affected tissues
C_LIO_LIMany aggregates that accumulate during physiological aging are linked to disease
C_LI


title: Pan-cancer proteomic map of 949 human cell lines reveals principles of cancer vulnerabilities

abstract: The proteome provides unique insights into biology and disease beyond the genome and transcriptome. Lack of large proteomic datasets has restricted identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types were analyzed by mass spectrometry. Deploying a clinically-relevant workflow to quantify 8,498 proteins, these data capture evidence of cell type and post-transcriptional modifications. Integrating multi-omics, drug response and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline revealed thousands of protein-specific biomarkers of cancer vulnerabilities. Proteomic data had greater power to predict drug response than the equivalent portion of the transcriptome. Further, random downsampling to only 1,500 proteins had limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger), available at https://cellmodelpassports.sanger.ac.uk, is a comprehensive resource revealing principles of protein regulation with important implications for future clinical studies.


title: High cardiomyocyte diversity in human early prenatal heart development

abstract:
Cardiomyocytes play key roles during cardiogenesis, but have poorly understood features, especially in prenatal stages. Thus, we have characterized human prenatal cardiomyocytes, 6.5- 7 weeks post-conception, in detail by integrating single-cell RNA sequencing, spatial transcriptomics, and ligand-receptor interaction information. Using a computational workflow developed to dissect cell type heterogeneity, localize cell types, and explore their molecular interactions, we identified eight types of developing cardiomyocyte, more than double compared to the ones identified in the Human Developmental Cell Atlas. These have high variability in cell cycle activity, mitochondrial content, and connexin gene expression, and are differentially distributed in the ventricles, including outflow tract, and atria, including sinoatrial node. Moreover, cardiomyocyte ligand-receptor crosstalk is mainly with non-cardiomyocyte cell types, encompassing cardiogenesis-related pathways. Thus, early prenatal human cardiomyocytes are highly heterogeneous and develop unique location-dependent properties, with complex ligand-receptor crosstalk. Further elucidation of their developmental dynamics may give rise to new therapies.

Graphical abstract

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title: Macrophage depletion blocks congenital SARM1-dependent neuropathy

abstract: Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified two rare NMNAT2 missense variants in two brothers afflicted with a progressive neuropathy syndrome. The polymorphisms result in amino acid substitutions, V98M and R232Q, which reduce NMNAT2 NAD+-synthetase activity. We generated a mouse model of the human syndrome and found that Nmnat2V98M/Nmnat2R232Q compound-heterozygous CRISPR mice survive to adulthood but develop progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes are all SARM1-dependent. Remarkably, macrophage depletion therapy blocks and reverses neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induces an inflammatory neuropathy and highlight the potential of immune therapy to treat this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

category: neuroscience


title: A novel lineage of RORγt+Aire+ antigen presenting cells promotes peripheral generation of intestinal regulatory T cells and tolerance during early life

abstract: Tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing Autoimmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. A second wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens within the first few weeks of life, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new lineage of tolerogenic ROR{gamma}t+ Aire+ antigen-presenting cells (APC) with a hybrid dendritic cell (DC)-mTEC phenotype, dubbed Thetis cells (TCs), comprising 4 major sub-groups (TC I-IV). We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While TC I bore remarkable homology with Aire+ mTECs, TC IV were specifically enriched for critical molecules required for pTreg generation, including the TGF-{beta} activating integrin v{beta}8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by ROR{gamma}t+ group 3 innate lymphoid cells (ILC3) and classical DCs was dispensable for pTreg generation, further implicating TCs as the critical tolerogenic ROR{gamma}t+ APC. Our studies reveal parallel pathways for establishment of tolerance within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.

category: immunology


title: Human MAIT cells show clonal diversity but transcriptional and functional homogeneity

abstract: Mucosal-associated invariant T (MAIT) cells are considered to have limited clonal diversity. In contrast, recent studies suggest the presence of functionally distinct subsets. We investigated this model through single-cell analysis of the MAIT cell TCR repertoire and transcriptional profile in human blood and liver. Further, we developed functional RNA-sequencing (fRNA-seq), an approach to integrate cellular function and TCR clonotype at a single-cell level following differential stimulation. MAIT cells showed surprising clonal diversity, with TCR repertoires shared across tissues but unique to individuals. Functional diversity within resting MAIT cells was low and largely related to tissue site. MAIT cells displayed distinct transcriptional responses to in vitro TCR and cytokine stimulation, with cells positioned along gradients of activation. Clonal origin influenced both resting and activated transcriptional profiles. Overall, MAIT cells exhibit diverse donor-specific TCR repertoires which, along with tissue and activation context, influence their phenotype and function.


title: HIF-1α induces glycolytic reprogramming in tissue-resident alveolar macrophagesto promote survival during acute lung injury

abstract: Cellular metabolism is a critical regulator of macrophage effector function. Tissue-resident alveolar macrophages (TR-AMs) inhabit a unique niche marked by high oxygen and low glucose. We have recently shown that in contrast to bone marrow-derived macrophages (BMDMs), TR-AMs cannot utilize glycolysis and predominantly rely on mitochondrial function for their effector response. It is not known how changes in local oxygen concentration that occur during conditions such as acute respiratory distress syndrome (ARDS) might affect TR-AM metabolism and function; however, ARDS is associated with progressive loss of TR-AMs, which correlates with the severity of disease and mortality. Here, we demonstrate that hypoxia robustly stabilizes HIF-1 in TR-AMs to promote a glycolytic phenotype. Hypoxia altered TR-AM metabolite signatures, cytokine production, and decreased their sensitivity to the inhibition of mitochondrial function. By contrast, hypoxia had minimal effects on BMDM metabolism. The effects of hypoxia on TR-AMs were mimicked by FG-4592, a HIF-1 stabilizer. Treatment with FG-4592 decreased TR-AM death and attenuated acute lung injury in mice. These findings reveal the importance of microenvironment in determining macrophage metabolic phenotype, and highlight the therapeutic potential in targeting cellular metabolism to improve outcomes in diseases characterized by acute inflammation.

category: cell biology


title: ATM modulates nuclear mechanics by regulating lamin A levels

abstract: Ataxia-telangiectasia mutated (ATM) is one of the three main apical kinases at the crux of DNA damage response and repair in mammalian cells. ATM activates a cascade of downstream effector proteins to regulate DNA repair and cell cycle checkpoints in response to DNA double-strand breaks. While ATM is predominantly known for its role in DNA damage response and repair, new roles of ATM have recently begun to emerge, such as in regulating oxidative stress or metabolic pathways. Here, we report the surprising discovery that ATM inhibition and deletion lead to reduced expression of the nuclear envelope protein lamin A. Lamins are nuclear intermediate filaments that modulate nuclear shape, structure, and stiffness. Accordingly, inhibition or deletion of ATM resulted in increased nuclear deformability and enhanced cell migration through confined spaces, which requires substantial nuclear deformation. These findings point to a novel connection between ATM and lamin A and may have broad implications for cells with ATM mutations--as found in patients suffering from Ataxia Telangiectasia and many human cancers--which could lead to enhanced cell migration and increased metastatic potential.


title: Realtime morphological characterization and sorting of unlabeled viable cells using deep learning

abstract:
Phenotyping of single cells has dramatically lagged advances in molecular characterization and remains a manual, subjective, and destructive process. We introduce COSMOS, a platform for phenotyping and enrichment of cells based on deep learning interpretation of high-content morphology data in realtime. By training models on an atlas of >1.5 billion images, we demonstrate enrichment of unlabeled cells up to 33,000 fold. We apply COSMOS to multicellular tissue biopsy samples demonstrating that it can identify malignant cells with similar accuracy to molecular approaches while enriching viable cells for functional evaluation. We show high-dimensional embedding vectors of morphology generated by COSMOS without any need for complex sample pre-processing, gating, or bioinformatics capabilities, which enables discovery of cellular phenotypes, and integration of morphology into multi-dimensional analyses.

One sentence summaryA novel platform capable of high-throughput imaging and gently sorting cells using deep morphological assessment.


title: A Pluripotent Developmental State Confers a Low Fidelity of Chromosome Segregation

abstract: Human pluripotent stem cells (hPSCs) frequently become aneuploid with abnormal chromosome numbers due to mitotic chromosome segregation errors during propagation in culture. Yet, we do not understand why hPSCs exhibit a low mitotic fidelity. Here we investigate the mechanisms responsible for mitotic errors in hPSCs and show that the primary cause is lagging chromosomes with improper merotelic chromosome microtubule attachments in anaphase. Accordingly, we can improve merotelic error correction and reduce lagging chromosome rates in hPSCs using small molecules that prolong mitotic duration or destabilize chromosome microtubule attachments providing chemical strategies to preserve genome stability. Strikingly, we also demonstrate that mitotic error rates correlate with developmental potential decreasing upon differentiation and loss of pluripotency and conversely increasing after reprogramming to a pluripotent state. Thus, chromosome segregation fidelity is inherently low in hPSCs and depends on developmental state in normal human cells.


title: BRAF-V600E-Mediated Erk Activation Promotes Sustained Cell Cycling and Broad Transcriptional Changes in Neonatal Cardiomyocytes

abstract: Mitogens capable of promoting cardiomyocyte proliferation represent important targets for functional heart regeneration following myocardial infarction. We previously described an ERK-dependent pro-proliferative tissue phenotype following overexpression of constitutively-active (ca) human ERBB2 in both neonatal rat ventricular myocytes (NRVMs) and human iPSC-derived cardiomyocytes (hiPSC-CMs). Since ERBB2 canonically regulates multiple other pathways in addition to ERK, it is unclear whether ERK activation alone can drive CM proliferation. Here, we activated ERK in a targeted fashion by CM-specific lentiviral expression of a constitutively active mutant of BRAF, BRAF-V600E (caBRAF), in cultured NRVMs and examined the effects on engineered NRVM tissue proliferation, morphology, and function. caBRAF expression induced ERK activation, tissue growth, loss of contractile function, and increased tissue stiffness, all of which were sustained for at least 4 weeks in vitro. From bulk RNA-sequencing analysis of engineered tissues, we found that caBRAF had broad transcriptomic effects on CMs and induced a shift to glycolytic metabolism. Together, this work shows that direct ERK activation is sufficient to modulate CM cycling and functional maturation in a cell-autonomous fashion and could offer a potential target for cardiac regenerative therapies.


title: Four novel, immortal cell lines derived from embryos of Xenopus tropicalis

abstract: The diploid anuran, Xenopus tropicalis, has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10, and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10, and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30{degrees}C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.


title: Nrf2 promotes thyroid development and hormone synthesis.

abstract: In all vertebrates, the function of the thyroid gland is to capture iodide to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Thyroid hormone production is a multistep process that ultimately leads to the coupling of iodine to thyroglobulin, the matrix glycoprotein of hormone synthesis. This coupling is dependent on H2O2, the accumulation of which in thyroid tissue, a true iodine trap, causes a basal level of oxidative stress higher than in other tissues, which, if left unchecked, leads to cellular damage. The thyroid has efficient antioxidant and detoxifying enzymes that help it resist H202-induced oxidative stress and maintain the homeostasis necessary for hormone synthesis. By regulating the expression of genes involved in cellular detoxification processes, the transcription factor NRF2 acts as a master regulator of the cellular defense system against oxidative stress. Using zebrafish embryos and mouse thyroid organoids, we show here that direct control of thyroglobulin expression by NRF2 is an evolutionarily conserved chore mechanism in vertebrates to complete the necessary molecular defense arsenal against oxidative stress in thyroid tissue.

category: developmental biology


title: microRNA-1 represses signaling pathway components to impact embryonic structures derived from all three germ layers

abstract:
microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. We use the sea urchin embryo to achieve a comprehensive understanding of miR-1s function in a developing embryo. Results indicate that miR-1 regulates gut contractions, specification, and positioning of serotonergic neurons, as well as mesodermally-derived muscles, pigment cells, and skeletogenic cells. Gain-of-function of miR-1 generally leads to more severe developmental defects than its loss-of-function. We identified that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Notch, Nodal, and Wnt1 signaling components. We found that miR-1s direct suppression of Nodal may indirectly regulate FoxQ2 to impact serotonergic neurons. Excess miR-1 may lead to decreased Nodal and Notch that result in decreased circumpharnygeal muscle fibers and the number of pigment cells. The striking ectopic skeletal branching induced by miR-1 mimic injections may be explained by miR-1s direct suppression of Nodal that leads to expression changes of Vegf3, and Fgfa that mediate skeletogenesis. This work demonstrates that miR-1 plays a diverse regulatory role that impacts tissues derived from all germ layers.

Summary statementThis study identifies wide-ranging regulatory roles and regulatory mechanisms of miR-1 that impact structures derived from all three germ layers during embryonic development.


title: When and where to count? Implications of migratory connectivity and non-breeding distribution to population censuses in a migratory bird population

abstract:
Migratory connectivity is a metric of the co-occurrence of migratory animals originating from different breeding sites, and like their spatio-temporal distributions, can vary substantially during the annual cycle. Together, both these properties affect the optimal times and sites of population censusing.

We tracked taiga bean geese (Anser fabalis fabalis) during 2014-2021 to study their migratory connectivity and non-breeding movements, and determine optimal periods to assess the size of their main flyway population. We also compared available census data with tracking data, to examine how well two existing censuses covered the population.

Daily Mantels correlation between breeding and non-breeding sites lay between 0 and 0.5 during most of the non-breeding season, implying birds from different breeding areas were not strongly separated other times in the annual cycle. However, the connectivity was higher among birds from the westernmost breeding areas compared to the birds breeding elsewhere. Daily Minimum Convex Polygons showed tracked birds were highly aggregated at census times, confirming their utility. The number of tracked birds absent at count sites during the censuses however exceeded numbers double-counted at several sites, indicating that censuses might have underestimated the true population size.

Our results show that connectivity can vary in different times during the non-breeding period, and should be studied throughout the annual cycle. Our results also confirm previous studies, which have found that estimates using marked individuals usually produce higher population size estimates than total counts. This should be considered when using total counts to assess population sizes in the future.


title: A pleiotropic flowering time QTL exhibits gene-by-environmental interaction for fitness in a perennial grass

abstract: Flowering time is crucial for wild plant populations to adapt to their local environments. Although the genetic basis of flowering variation has been studied in many plant species, its mechanisms in non-model organisms and its adaptive value in the field are still poorly understood. Here, we report new insights into the genetic basis of flowering time and its effect on fitness in Panicum hallii, a native perennial grass. We conducted genetic mapping in populations derived from representative inland and coastal ecotypes to identify flowering time QTL and loci exhibited extensive QTL-by-environment interactions. Patterns of segregation within recombinant hybrids provide strong support for directional selection driving ecotypic divergence in flowering time. A major QTL on chromosome 5 (q-FT5) was detected in all experiments and is a key locus controlling flowering variation. Fine-mapping and expression studies identified a FLOWERING LOCUS T orthologue, FT-like 9 (PhFTL9), as the candidate underlying q-FT5. We used reciprocal transplant experiment to test for global local adaptation and the specific impact of q-FT5 on performance. We did not observe local adaptation in terms of fitness tradeoffs when contrasting ecotypes in home versus away habitats. However, we observed that the coastal allele of q-FT5 conferred a fitness advantage only in its local habitat but not at the inland site. Sequence analysis of the PhFTL9 promoter identified ecotypic specific cis-element variation associated with environmental responsiveness. Together, our findings demonstrate the genetic basis of flowering variation in a perennial grass and provide evidence for conditional neutrality underlying flowering divergence.

category: evolutionary biology


title: MoIsw2 activity could be the missing link between adaptation and mutation instrumental for natural adaptation-directed fast evolution (NADFE) of Magnaporthe oryzae

abstract:
Isw2 is known to bind DNA, using a Myb-type DNA binding domain and with another part of the protein interact with histones in the closest nucleosome to pull the two closest nucleosomes closer to the binding site using ATP. These bindings hinder these nucleosomes sliding movements and affect histone tail modification and DNA methylation but give more space for sliding and access for transcription factors further away from the Isw2 DNA binding sites. In a previous paper, we have knocked out the Magnaporthe oryzae Isw2 protein and complemented it with a GFP-labelled construct. We found that the MoISW2 is needed, especially in the later necrotrophic stages of infection when the plant starts defending itself.

Using published transcriptomic data, we have now found that MoISW2 is mainly co-regulated with Histone 4 (MoHIS4), which likely indicates that MoIsw2 predominately interacts with MoHis4. We performed a ChIP-seq experiment using MoIsw2-GFP to map the binding sites and investigated if MoIsw2 regulates genes by the limited sliding type and if this regulation could be involved in pathogenicity regulation. Common motifs were found in the binding DNA sequences using MEME. Interestingly, these motifs were mainly intergenic, and a dominant palindrome motif was present in 200 sequences.

We further performed an RNAseq for{Delta} Moisw2 compared with Ku80 background strain and used genes sorted in physical order on the supercontigs to determine the effect of the mutation on the regulation of the genes physically close to the MoIsw2 binding sites. The genes close to the binding site were more absolutely regulated in the{Delta} Moisw2 mutant. As nucleosomes are evenly distributed at roughly 200bp distance, there should be an interference pattern of regulation of genes centered around the MoIsw2 DNA binding site if the genes are roughly evenly distributed, which could be confirmed. We queried the NCBI database again and checked where the 200 palindromic sequences were found in the genome to investigate if MoIsw2 binds to any known structures in the DNA. The palindromic sequences were found in conserved retrotransposon elements that generally contain palindromes. Since these retrotransposon elements can move place, they can create new regulatory variations with MoIsw2. Many avirulence genes recognized by different host plant cultivars and typically vary in expression between pathogen isolates were close to the MoIsw2 DNA binding site. That could mean that mobile elements associated with some MoIsw2 DNA binding sites are involved in creating variations in the pathogenicity of different M. oryzae strains. To test this idea, we searched through the transcriptomic data of our strain (70-15) and another strain (98-05) for variation in AVR expression during infection and if differences in MoIsw2 binding could be the cause for this variation and it seems to be that. Finally, we found that most of the genes upregulated in the{Delta} Moisw2 mutant regulate biomass growth (DNA-binding and quality control and TFs overrepresented). In contrast, the ones downregulated have to do with mainly mitochondrial oxidative phosphorylation, oxidative stresses, and secondary metabolites. Similar results for enriched and depleted genes were found for all genes in our ChIP-seq data for genes closest to MoIsw2 binding. We conclude that MoIsw2 interaction with nucleosomes and binding to palindromic DNA in transposons located between genes creates a possibility for fast variation of offspring regulatory pattern without loss of genes through mutation and fast creation of M.oryzae strains with new cultivar host ranges in response to defenses by resistant cultivars and consequently a fast break of resistance. MoISW2 also seems to aid in the natural adaptation-directed fast evolution (NADFE) of M. oryzae, which is discussed as a new concept for Eukaryotes.


title: Interactions of C. elegans -tubulins with the microtubule inhibitor albendazole

abstract: Parasitic nematodes are major human and agricultural pests, and benzimidazoles are amongst the most important broad spectrum anthelmintic drug class used for their control. Benzimidazole resistance is now widespread in many species of parasitic nematodes in livestock globally and an emerging concern for the sustainable control of human soil transmitted helminths. {beta}-tubulin is the major benzimidazole target, although other genes may influence resistance. Among the six C. elegans {beta}-tubulin genes, loss of ben-1 causes resistance without other apparent defects. Here, we explored the genetics of C. elegans {beta}-tubulin genes in relation to the response to the benzimidazole derivative albendazole. The most highly expressed {beta}-tubulin isotypes, encoded by tbb-1 and tbb-2, were known to be redundant with each other for viability, and their products are predicted not to bind benzimidazoles. We found that tbb-2 mutants, and to a lesser extent tbb-1 mutants, were hypersensitive to albendazole. The double mutant tbb-2 ben-1 is uncoordinated and dumpy, resembling the wild type exposed to albendazole, but the tbb-1 ben-1 double mutant did not show the same phenotype. These results suggest that tbb-2 is a modifier of ABZ sensitivity. To better understand how BEN-1 mutates to cause benzimidazole resistance, we isolated mutants resistant to albendazole and found that 15 of 16 mutations occurred in ben-1. Mutations ranged from likely nulls to hypomorphs, and several corresponded to residues that cause resistance in other organisms. Null alleles of ben-1 are albendazole-resistant and BEN-1 shows high sequence identity with tubulins from other organisms, suggesting that many amino acid changes could cause resistance. However, our results suggest that missense mutations conferring resistance are not evenly distributed across all possible conserved sites. Independent of their roles in benzimidazole resistance, tbb-1 and tbb-2 may have specialized functions as null mutants of tbb-1 or tbb-2 were cold or heat sensitive, respectively.

category: genetics


title: Environmental DNA reflects common haplotypic variation

abstract: Analysis of environmental DNA (eDNA) has gained widespread usage for taxonomically based biodiversity assessment. While interest in applying non-invasive eDNA monitoring for population genetic assessments has grown, its usage in this sphere remains limited. One barrier to uptake is that the effectiveness of eDNA detection below the species level remains to be determined for multiple species and environments. Here, we test the utility of this emergent technology in a population genetic framework using eDNA samples derived from water along New Zealands South Island (Otago Coast: n=9; Kaik[o]ura: n=7) and DNA obtained from tissue samples (n=76) of individual blackfoot p[a]ua (Haliotis iris) sampled from New Zealands Otago coast. We recovered four mitochondrial haplotypes from eDNA versus six from the tissue samples collected. Three common haplotypes were recovered with both eDNA and tissue samples, while only one out of three rare haplotypes - represented in tissue samples by one individual each - was recovered with our eDNA methods. We demonstrate that eDNA monitoring is an effective tool for recovering common genetic diversity from p[a]ua, although rare (< 5%) haplotypes are seldom recovered. Our results show the potential of eDNA to identify population-level haplotypes for gastropods in the marine environment identification below the species level and for studying the population genetic diversity of gastropods. This work supports eDNA methods as effective, non-invasive tools for genetic monitoring. Non-invasive eDNA sampling could minimize target organism stress and human interaction enabling population genetic research for hard-to-sample, delicate, or sensitive species.


title: A clinically annotated post-mortem approach to study multi-organ somatic mutational clonality in histologically healthy tissues.

abstract: Recent research on histologically healthy human tissues identified omnipresent mutational microclones, driven by somatic mutations known to be responsible for carcinogenesis (e.g., in TP53 or NOTCH1). These new insights are fundamentally changing current tumour evolution models, with broad oncological implications. Most studies are based on surgical remnant tissues, which are not available for many organs and rarely in a pan-organ setting (multiple organs from the same individual). Here, we describe an approach based on clinically annotated post-mortem tissues, derived from whole-body donors that are routinely used for educational purposes at human anatomy units. We validated this post-mortem approach using UV-exposed and unexposed epidermal skin tissues and confirm the presence of positively selected NOTCH1/2-, TP53- and FAT1-driven clones. No selection signals were detected in a set of immune genes or housekeeping genes. Additionally, we provide the first evidence for smoking-induced clonal changes in oral epithelia, likely underlying the origin of head and neck carcinogenesis. In conclusion, the whole-body donor-based approach provides a nearly unlimited healthy tissue resource to study mutational clonality and gain fundamental mutagenic insights in the presumed earliest stages of tumour evolution.

category: genomics


title: Assessing temporal and geographic contacts across the Adriatic Sea through the analysis of genome-wide data from Southern Italy

abstract: Southern Italy was characterised by a complex prehistory that started with different Palaeolithic cultures, later followed by the Neolithic transition and the demic dispersal from the Pontic-Caspian Steppe during the Bronze Age. Archaeological and historical evidence points to demic and cultural influences between Southern Italians and the Balkans, starting with the initial Palaeolithic occupation until historical and modern times. To shed light on the dynamics of these contacts, we analysed a genome-wide SNP dataset of more than 700 individuals from the South Mediterranean area (102 from Southern Italy), combined with ancient DNA from neighbouring areas. Our findings revealed high affinities of South-Eastern Italians with modern Eastern Peloponnesians, and a closer affinity of ancient Greek genomes with those from specific regions of South Italy than modern Greek genomes. The higher similarity could be associated with the presence of a Bronze Age component ultimately originating from the Caucasus and characterised by high frequencies of Iranian and Anatolian Neolithic ancestries. Furthermore, to reveal possible signals of natural selection, we looked for extremely differentiated allele frequencies among Northern and Southern Italy, uncovering putatively adapted SNPs in genes involved in alcohol metabolism, nevi features and immunological traits, such as ALDH2, NID1 and CBLB.


title: MemBrain: A Deep Learning-aided Pipeline for Automated Detection of Membrane Proteins in Cryo-electron Tomograms

abstract:
Background and ObjectiveCryo-electron tomography (cryo-ET) is an imaging technique that enables 3D visualization of the native cellular environment at sub-nanometer resolution, providing unpreceded insights into the molecular organization of cells. However, cryo-electron tomograms suffer from low signal-to-noise ratios and anisotropic resolution, which makes subsequent image analysis challenging. In particular, the automated detection of membrane-embedded proteins is a problem still lacking satisfactory solutions.

MethodsWe present MemBrain - a new deep learning-based pipeline that automatically detects membrane-bound protein complexes in cryo-electron tomograms. After subvolumes are sampled along a segmented membrane, each subvolume is assigned a score using a convolutional neural network (CNN), and protein positions are extracted by a clustering algorithm. Incorporating rotational subvolume normalization and using a tiny receptive field simplify the task of protein detection and thus facilitate the network training.

ResultsMemBrain requires only a small quantity of training labels and achieves excellent performance with only a single annotated membrane (F1 score: 0.88). A detailed evaluation shows that our fully trained pipeline outperforms existing classical computer vision-based and CNN-based approaches by a large margin (F1 score: 0.92 vs. max. 0.63). Furthermore, in addition to protein center positions, MemBrain can determine protein orientations, which has not been implemented by any existing CNN-based method to date. We also show that a pre-trained MemBrain program generalizes to tomograms acquired using different cryo-ET methods and depicting different types of cells.

ConclusionsMemBrain is a powerful and label-efficient tool for the detection of membrane protein complexes in cryo-ET data, with the potential to be used in a wide range of biological studies. It is generalizable to various kinds of tomograms, making it possible to use pretrained models for different tasks. Its efficiency in terms of required annotations also allows rapid training and fine-tuning of models. The corresponding code, pretrained models, and instructions for operating the MemBrain program can be found at: https://github.com/CellArchLab/MemBrain


title: Reconstructing the evolutionary history of a BCR lineage with minimum spanning tree and clonotype abundances

abstract: MotivationB cell receptor (BCR) genes exposed to an antigen undergo somatic hypermutations and Darwinian antigen selection, generating a large BCR-antibody diversity. This process, known as B cell affinity maturation, increases antibody affinity, forming a specific B cell lineage that includes the unmutated ancestor and mutated variants. In a B cell lineage, cells with a higher antigen affinity will undergo clonal expansion, while those with a lower affinity will not proliferate and probably be eliminated. Therefore, the abundance of different genotypes provides a valuable perspective on the ongoing evolutionary process. Phylogenetic tree inference is often used to reconstruct B cell lineage trees and represents the evolutionary dynamic of BCR affinity maturation. However, such methods should process B cell population data derived from experimental sampling that might contain different cellular abundances. There are a few phylogenetic methods for reconstructing the evolutionary history of B cell lineages; best-performing solutions are time-demanding and restricted to analyze a reduced number of BCR sequences, while time-efficient methods do not consider cellular abundances. We propose ClonalTree, a low-complexity and accurate approach to reconstruct BCR lineage trees that incorporates genotype abundances into minimum spanning tree (MST) algorithms. Using both simulated and experimental data, we demonstrated that ClonalTree outperforms MST-based algorithms and achieves a similar performance compared to a method that explores tree generating space exhaustively. However, ClonalTree has a lower running time, being more convenient for reconstructing phylogenetic lineage trees from high-throughput BCR sequencing data, mainly in biomedical applications, where a lower computational time is appreciable. It is hundreds to thousands of times faster than exhaustive approaches, enabling the analysis of a large set of sequences within minutes or seconds and without loss of accuracy. The source code is freely available at github.com/julibinho/ClonalTree.


title: Single-cell RNA Sequencing of Pediatric Ependymoma Unravels Subclonal Heterogeneity Associated with Patient Survival

abstract: Ependymoma (EPN) is a malignant glial tumor occurring throughout central nervous system which commonly presents in children. Although recent studies have characterized EPN samples at both the bulk and single-cell level, intra-tumoral heterogeneity across subclones remains a confounding factor which impedes understanding of EPN biology. In this study, we generated a high-resolution single-cell dataset of pediatric ependymoma with a particular focus on the comparison of subclone differences within tumors, and show upregulation of cilium-associated genes in more highly differentiated subclone populations. As a proxy to traditional pseudotime analysis, we applied a novel trajectory scoring method to reveal cellular compositions associated with poor survival outcomes across primary and relapsed patients. Furthermore, we identified putative cell-cell communication features between relapsed and primary samples and show upregulation of pathways associated with immune cell crosstalk. Our results reveal both inter- and intratumoral gene expression profiles and tumor differentiation and provide a framework for studying transcriptomic signatures of individual subclones in ependymoma at single-cell resolution.


title: Development of Dry Powder Formulations of AS01B containing vaccines using Thin-Film Freeze-Drying

abstract: AS01B is a liposomal formulation of two immunostimulants namely 3-O-desacyl-4-monophosphoryl lipid A (MPL) and QS-21. The liposomal formulation of AS01B reduces the endotoxicity of MPL and the lytic activity of QS-21; however, it renders the adjuvant sensitive to accidental slow freezing. The liposomal formulation also represents a major challenge towards the formulation of dry powders of vaccines containing AS01B. In the present study, we tested the feasibility of applying thin-film freeze-drying (TFFD) to engineer dry powders of the AS01B liposomal adjuvant alone or vaccines containing AS01B as an adjuvant. Initially, we showed that after the AS01B liposomal adjuvant was subjected to TFFD using sucrose as a stabilizer at 4% w/v, the particle size distribution of AS01B liposomes reconstituted from the dry powder was identical to the liquid adjuvant before drying. We then showed using ovalbumin (OVA) as a model antigen adjuvanted with AS01B (AS01B/OVA) that subjecting the AS01B/OVA vaccine to TFFD and subsequent reconstitution did not negatively affect the AS01B liposome integrity, nor the immunogenicity of the vaccine. Importantly, the thin-film freeze-dried vaccine was not sensitive to repeated freezing-and-thawing. Finally, the feasibility of using TFFD to prepare dry powders of AS01B-adjuvanted vaccines was further confirmed using AS01B-adjuvanted Fluzone Quadrivalent and Shingrix, which contains AS01B. It is concluded that the TFFD technology can enable the formulation of AS01B-adjuvanted vaccines as freezing-insensitive dry powders in single-vial presentation.


title: CLE42 Delays Leaf Senescence by Antagonizing Ethylene Pathway in Arabidopsis

abstract: Leaf senescence is the final stage of leaf development and is influenced by numerous internal and environmental factors. CLE family peptides are plant-specific peptide hormones that regulate various developmental processes. However, the role of CLE in regulating leaf senescence remains unclear. Here, we found that CLE42 is a negative regulator of leaf senescence by using a CRISPR/Cas9-produced CLE mutant collection. The cle42 mutant displayed earlier senescence phenotypes, while overexpression of CLE42 delayed age-dependent and dark-induced leaf senescence. Moreover, application of the synthesized 12-aa peptide (CLE42p) also delayed leaf senescence under natural and dark conditions. CLE42 and CLE41/44 displayed functional redundancy in leaf senescence, and the cle41 cle42 cle44 triple mutant displayed more pronounced earlier senescence phenotypes than any single mutant. Analysis of differentially expressed genes obtained by RNA-Seq methodology revealed that ethylene pathway was suppressed by overexpressing CLE42. Moreover, CLE42 suppressed ethylene biosynthesis and thus promoted the protein accumulation of EBF, which in turn decreased the function of EIN3. Accordingly, mutation of EIN3/EIL1 or overexpression of EBF1 suppressed the earlier senescence phenotypes of the cle42 mutant. Together, our results reveal that the CLE peptide hormone regulates leaf senescence by communicating with ethylene pathway.


title: Drastic differential CRISPR-Cas9 induced mutagenesis influenced by DNA methylation and chromatin features

abstract: CRISPR-Cas9 mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies considered DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence revealed the significant role of chromatin context. Nonetheless, most of the prior studies were limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis thaliana to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to significant variations in mutagenesis efficiency by up to 250 folds. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variations in CRISPR-Cas9 mutation profiles reflected by the 1 bp insertion rates. Our findings provided strong evidence that specific chromatin features could have significant and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double strand break repair outcomes.


title: PhD and postdoc training outcomes at EMBL: changing career paths for life scientists in Europe.

abstract: The life sciences are training growing numbers of PhDs and postdocs, who increasingly engage in collaborative research. The impact of these changes on the careers of researchers is, however, unclear. Here, we report an analysis of the training outcomes for 2284 researchers who completed a PhD or postdoc at the European Molecular Biology Laboratory (EMBL) between 1997 and 2020. This is the first such study published from a European institute and first time-resolved analysis globally. The most common career outcomes were in academic research, service and teaching (1263 alumni, 55%), including 636 principal investigators (PIs). A broad spectrum of other career paths was also represented, including in industry research (332, 15%) and science-related professions (349, 15%). Our analysis indicates that, although there is increased competition for PI roles, life scientists continue to enter and excel in careers that drive research and innovation.

category: scientific communication and education


title: Local Subspace Pruning (LSP) for Multichannel Data Denoising

abstract: This paper proposes a simple algorithm to remove noise and artifact from multichannel data. Data are processed trial by trial: for each trial the covariance matrix of the trial is diagonalized together with that of the full data to reveal the subspace that is - locally - most eccentric relative to other trials. That subspace is then projected out from the data of that trial. This algorithm addresses a fundamental limitation of standard linear analysis methods (e.g. ICA) that assume that brain and artifact are linearly separable within the data. That assumption fails if there are more sources, including noise and brain sources, than data channels. The algorithm captitalizes on the fact that, if enough of those sources are temporally sparse, linear separation may succeed locally in time. The paper explains the rationale, describes the algorithm, and evaluates the outcome using synthetic and real brain data.


title: Identification of protein aggregates in the aging vertebrate brain with prion-like andphase separation properties

abstract: Protein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during normal brain aging remains unknown. Here we use quantitative proteomics in the African turquoise killifish to identify protein aggregates that accumulate in old vertebrate brains. These aggregates are enriched for prion-like RNA binding proteins, notably the ATP-dependent RNA helicase DDX5. We validate that DDX5 forms mislocalized cytoplasmic aggregates in the brains of old killifish and mice. Interestingly, DDX5s prion-like domain allows these aggregates to propagate across many generations in yeast. In vitro, DDX5 phase separates into condensates. Mutations that abolish DDX5 prion propagation also impair the proteins ability to phase separate. DDX5 condensates exhibit enhanced enzymatic activity, but they can mature into inactive, solid aggregates. Our findings suggest that protein aggregates with prion-like properties form during normal brain aging, which could have implications for the age-dependency of cognitive decline.


title: The laminin-keratin link shields the nucleus from mechanical deformation and signalling

abstract: The mechanical properties of the extracellular matrix (ECM) dictate tissue behaviour. In epithelial tissues, laminin is both a very abundant ECM component, and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111, unlike fibronectin or collagen I, impairs cell response to substrate rigidity, and YAP nuclear localization. Blocking the laminin-specific integrin {beta}4 increases nuclear YAP ratios in a rigidity dependent manner, without affecting cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that {beta}4 integrins establish a mechanical linkage between the substrate and the keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects nuclear YAP mechanoresponses and chromatin methylation. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.


title: Anatomical structures, cell types, and biomarkers of the healthy human blood vasculature

abstract: More than 150 scientists from 17 consortia are collaborating on an international project to build a Human Reference Atlas, which maps all 37 trillion cells in the healthy adult human body. The initial release of this atlas provided hierarchical lists of the anatomical structures, cell types, and biomarkers in 11 organs. Here, we describe the methods we used as part of this initiative to build the first open, computer-readable, and comprehensive database of the adult human blood vasculature, called the Human Reference Atlas-Vasculature Common Coordinate Framework (HRA-VCCF). It includes 961 vessels and their branching connections, 10 cell types, and 10 biomarkers. With this paper we are releasing additional details on vessel types and subtypes, branching sequence, anastomoses, portal systems, microvasculature, functional tissue units, mappings to regions vessels supply or drain, and links to 3D reference objects. Future versions will add variants, geometric properties of vessels, and connections to the lymph vasculature; and, it will iteratively expand and improve the database as additional experimental data become available through the participating consortia.


title: Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration

abstract: Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays important roles in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of {Delta}Fgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.


title: IGF2BP1/IMP1 contributes to autophagy modulation directly via MAP1LC3B

abstract: Homeostatic tissue maintenance requires coordinated regulation of metabolic processes including macroautophagy/autophagy. Autophagy dysregulation underlies numerous human diseases. Our prior work revealed that the RNA binding protein IGF2BP1/IMP1 binds transcripts encoding autophagy-related proteins. Furthermore, Imp1 deletion in gastrointestinal epithelial cells in mice was associated with enhanced autophagy flux and improved recovery from tissue injury. In the current study, we evaluated molecular mechanisms underlying IMP1 modulation of autophagy. We report increased expression of autophagy protein microtubule associated protein 1 light-chain 3B (MAP1LC3B) in cells with IMP1 deletion compared to control cells. MAP1LC3B expression was also increased in cells with mutated IMP1 phosphorylation sites. Luciferase reporter assays demonstrate increased translation of MAP1LC3B-3UTR in cells with IMP1 deletion. Furthermore, we find that IMP1 co-localizes with MAP1LC3B transcripts at homeostasis, and co-localization is decreased in cells where IMP1 phosphorylation sites are mutated. IMP1 localization with MAP1LC3B transcripts is also decreased following cell stress. Taken together, our data support a model whereby IMP1 regulates MAP1LC3B translation which can serve as a mechanism to calibrate autophagy levels in the cell. This new mechanism may be particularly important in gastrointestinal epithelial cells, where autophagy is essential for tissue recovery following injury, or in diseases such as inflammatory bowel disease where defective autophagy is implicated.


title: Insights into the role of Nup62 and Nup93 in assembling cytoplasmic ring and central transport channel of the nuclear pore complex

abstract: The nuclear pore complex (NPC) is a highly modular assembly of 34 distinct nucleoporins (Nups), to form a versatile transport channel between the nucleus and cytoplasm. Among them, Nup62 is known as an essential component for nuclear transport while, Nup93 for the proper nuclear envelope assembly. These Nups constitute various NPC subcomplexes: such as central transport channel (CTC), cytoplasmic ring (CR) and inner ring (IR). However, how they play their role in the NPC assembly and transport activity is not clear. Here we delineated the interacting regions, conducted biochemical reconstitution and structural characterization of the mammalian CR complex to reveal its intrinsic dynamic behaviour and a distinct  4 shaped architecture resembling the CTC complex. Our data demonstrate that Nup62 coiled-coil domain is critical to form both Nup62*Nup88 and Nup62*Nup88*Nup214 heterotrimers and both can bind to the Nup93. We therefore propose that Nup93 act as a  sensor to bind to Nup62 shared heterotrimers including Nup62*Nup54 heterotrimer of the CTC, which was not shown previously as an interacting partner. Altogether, our study establishes that the Nup62 via its coiled-coil domain is central to form compositionally distinct yet structurally similar heterotrimers, and the Nup93 anchors these diverse heterotrimers by recognizing them non-selectively.

category: biochemistry


title: In utero delivery of miRNA induces epigenetic alterations and corrects pulmonary pathology in congenital diaphragmatic hernia

abstract:
Structural fetal diseases, such as congenital diaphragmatic hernia (CDH) can be diagnosed prenatally. Neonates with CDH are healthy in utero as gas exchange is managed by the placenta, but impaired lung function results in critical illness from the time a baby takes its first breath. During fetal development, lungs are capable of remarkable growth and the fetus does not yet require lung function for gas exchange. MicroRNA (miR) 200b and its downstream targets in the TGF{beta} pathway are critically involved lung branching morphogenesis. Here we characterize the expression of miR200b and the TGF{beta} pathway at different gestational times using a rat model of CDH. Fetal rats with CDH are deficient in miR200b at gestational day 18. We demonstrate that NPs loaded with miR200b given systemically to fetal rats result in changes in the TGF{beta} pathway; these epigenetic changes improve lung size, lung morphology, and lung vascularization. This is the first demonstration of in utero epigenetic therapy to improve lung growth and development in a pre-clinical model. With refinement, this technique could be applied to fetal cases of CDH or other forms of impaired lung development in a minimally invasive fashion.

eTOC SynopsisIn utero treatment with NPs loaded with miR200b improves lung development in a rat model of CDH. miR200b treatment epigenetic changes in the TGF{beta}, leads to larger lungs with more airspace and favorable pulmonary vascular remodeling.

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title: Acinetobase: the comprehensive database and repository of Acinetobacter strains.

abstract:
BackgroundAcinetobacter baumannii is one of the most problematic nosocomial pathogens that can efficiently thrive within hospital settings, mainly due to resistances towards antibiotics, desiccation, disinfectants, human serum and oxidative stress. Recently, increased resistance against last resort antibiotics earns this bacterium the highest priority concern classified by the CDC and the WHO. An obvious hallmark of this bacterium is the high heterogeneity observed amongst A. baumannii isolates, with a limited core genome. This feature complexifies the study of A. baumannii bacteria as an entity, subsequently reflected in a diversity of phenotypes of not only antimicrobial and environmental resistance but also virulence.

Main bodyHigh degree of genome plasticity, along with the use of a limited subset of established reference strains, can lead to strain-specific observations, decreasing the global understanding of this pathogenic agent. Phenotypic variability of A. baumannii strains is easily observable such as with the macrocolony morphologies, in vitro and in vivo virulence, natural competence level, production of different capsular polysaccharide structures and cellular densities. Some strains encode an extensive amount of virulence factors while other, including the reference strains, lack several key ones. The lack/excess of genes or specific physiological processes might interfere with in vivo and in vitro experiments, thus providing limited impact on the global understanding of Acinetobacter bacteria as a whole.

ConclusionAs an answer to the high heterogeneity amongst A. baumannii strains, we propose a first comprehensive database that includes the bacterial strains and the associated phenotypic and genetic data. This new repository, freely accessible to the entire scientific community, allow selecting the best bacterial isolate(s) related to any biological question, using an efficient and fast exchange platform.


title: A2A Receptor contributes to tumor progression in P2X7 null mice

abstract: ATP and adenosine are key constituents of the tumor niche where they exert opposite and complementary roles. ATP promotes tumor growth but also immune eradicating responses mainly via the P2X7 receptor (P2X7R), while adenosine acts as a potent immune suppressor and facilitates neovascularization thanks to A2A receptor (A2AR) activity. However, studies exploring the interplay between P2X7R and A2AR in the tumor microenvironment are as yet missing. Here we investigated tumor growth in C57/bl6 P2X7 null mice inoculated with B16-F10 melanoma cells, showing that several pro-inflammatory cytokines (IL1-{beta}, TNF-, IL-6, IL-12, IL-17, IFN-{gamma}) were significantly decreased while the immune suppressant TGF-{beta} was almost three-fold increased. Interestingly, tumors growing in P2X7-null mice also upregulated tumor-associated and splenic A2AR, suggesting that immunosuppression associated to lack of the P2X7R might depend upon A2AR overexpression. Immunohistochemical analysis showed that tumor cells A2AR expression was increased, especially around necrotic areas, and that VEGF and the endothelial marker CD31 were also upregulated. The A2AR antagonist SCH-58261 reduced tumor growth similarly in the P2X7 WT, or null mice strain. However, SCH-58261only reduced VEGF in the P2X7-KO mice, thus supporting the hypothesis of an A2AR mediated increase in vascularisation in P2X7-null host. SCH-58261 administration also significantly reduced intratumor TGF-{beta}, thus supporting a key immune suppressive role of A2AR in our model. This study shows a novel direct correlation between P2X7R and A2AR in oncogenesis and paves the way for new combined therapies promoting anti-cancer immune responses and reducing tumor vascularization.

category: cancer biology


title: Assessment of dynamics of global DNA methylation during the cryopreservation process of Pinus pinaster embryogenic lines

abstract: Long-term in vitro maintenance of embryogenic lines of Pinus species has been associated with lower maturation capacity, because of this, cryopreservation protocols for the embryogenic lines are needed to maintain valuable genotypes. Since cryopreservation may induce epigenetic variations, we evaluate changes in DNA methylation levels through the course of the cryopreservation of maritime pine embryogenic lines, as compared to those lines maintained by repeated subcultures. Six maritime pine embryogenic lines were cryopreserved following a protocol that includes pre-treatments inducing osmotic stress in liquid media. The percentage of methylated cytosines (%5-mC) in total DNA was determined by using a colorimetric assay. Reactive oxygen species (ROS) accumulation in cell lines was also determined by quantifying dihydroethidium (DHE) fluorescence under a confocal laser-scanning microscope. In the first experiment, we found that global DNA methylation was significantly reduced during the cryopreservation protocol. Subsequently, we evaluated the methylation status of both cryopreserved and no cryopreserved lines (maintained by subcultures) and found differences among embryogenic lines but overall, cryopreservation did not alter %5-mC of the recovered lines while periodical subcultures increased methylation rates. In addition, maltose pretreatment did not increase significantly ROS production in embryogenic lines. Our results demonstrate that the genetic stability during cryopreservation highly depends on the embryogenic line studied, but the protocol allows maintaining methylation DNA rates in most of the recovered lines.


title: Social cues of safety can override differences in threat level

abstract: Animals in groups integrate social information with that directly-gathered about the environment to guide decisions regarding reproduction, foraging and defense against predatory threats. In the context of predation, usage of social information has acute fitness benefits, aiding the detection of predators, the mounting of concerted defensive responses, or allowing the inference of safety, permitting other beneficial behaviors such as foraging for food. Individual and group defense responses to predatory threats can vary in modality and vigor depending on the perceived threat level. Moreover, predation level has been shown to modulate the use of social cues about foraging sites. Whether and how different threat levels affect the use of social cues to guide defense responses, is currently unknown. We previously showed that Drosophila melanogaster display a graded decrease in freezing behavior, triggered by an inescapable visual threat, with increasing group sizes. Crucially, we identified the movement of others as a cue of safety and its cessation a cue of threat and found the group responses to be primarily guided by the safety cues, resulting in a net social buffering effect. Here, we investigated how threat level impacts the use of social cues by exposing flies individually and in groups to two threat imminences using looms of different speeds. We show that freezing responses are stronger to the faster looms regardless of social condition. However, social buffering was stronger for groups exposed to the fast looms, such that the increase in freezing caused by the higher threat was less prominent in flies tested in groups than those tested individually. Through artificial control of behavior, we created different group compositions, titrating the motion cues that were maintained across threat levels. We, found that the same level of safety motion cues had a bigger weight on the flies decisions when these were exposed to the higher threat, thus overriding differences in perceived threat levels. These findings shed light on the  safety in numbers effect, revealing the modulation of the saliency of social safety cues across threat intensities, a possible mechanism to regulate costly defensive responses.


title: Sex differences in contextual pattern separation, activation of new neurons, and functional connectivity within the limbic system

abstract: Sex differences in the structure and function of hippocampus exist. Here, we examined sex differences in contextual pattern separation, functional connectivity, and activation of new neurons during fear memory. Two-month-old male and female Sprague-Dawley rats were injected with the DNA synthesis markers, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) three weeks and four weeks before perfusion, respectively. One week after CldU injection, the rats underwent a context discrimination task in which rats were placed in context A (shock) and context A (no shock) every day for 12 days. On the test day, rats were placed in the shock context (context A) to measure fear memory and expression of zif268, an immediate early gene across 15 different limbic and reward regions. We found that females, but not males, showed contextual discrimination during the last days of training. On the test day, both sexes displayed similar levels of freezing, indicating equivalent fear memory for context A. Despite similar fear memory, males showed more positive correlations of zif268 activation between the limbic regions and the striatum, whereas females showed more negative correlations among these regions. Females showed greater activation of the frontal cortex, dorsal CA1, and 3-week-old adult-born dentate granular cells compared to males. These results highlight the importance of studying sex differences in fear memory and the contribution of adult neurogenesis to the neuronal network and may contribute to differences in susceptibility to fear related disorders such as posttraumatic stress disorder.


title: Increased reliability of visually-evoked activity in area V1 of the MECP2-duplication mouse model of autism

abstract: Atypical sensory processing is now thought to be a core feature of the autism spectrum. Influential theories have proposed that both increased and decreased neural response reliability within sensory systems could underlie altered sensory processing in autism. Here, we report evidence for abnormally increased reliability of visual-evoked responses in layer 2/3 neurons of adult primary visual cortex in the MECP2-duplication syndrome animal model of autism. Increased response reliability was due in part to decreased response amplitude, decreased fluctuations in endogenous activity, and decreased neuronal coupling to endogenous activity. Similarly to what was observed neuronally, the optokinetic reflex occurred more reliably at low contrasts in mutant mice compared to controls. Retinal responses did not explain our observations. These data suggest that the circuit mechanisms for convolution of sensory-evoked and endogenous signal and noise may be altered in this form of syndromic autism.


title: Koisio Technology-Modulated Cell Culture Media can Lead to Markedly Increased Intracellular ATP Levels of Fibroblast Cell Cultures

abstract: It has been reported that Koisio technology-modulated water without external additions of any substances can enter several types of aquaporins with significantly higher rate. In this study we determined the effects of Koisio technology-modulated cell culture media (Koisio media) on the energy state of cells. We found that for the lung fibroblast (L929) cell cultures, Koisio media without any additions of external substances led to approximately an 57% increase in the intracellular ATP levels of the cell cultures at the Cell Passage 6 - 18, compared with regular cell culture media. Seemingly surprisingly, we did not find that Koisio media could significantly affect the mitochondrial membrane potential of the cells, compared with regular cell culture media. Collectively, our study has provided first evidence indicating that Koisio media can lead to remarkable increases in the intracellular ATP levels of the L929 cell cultures, suggesting significant potential of Koisio technology in generating energy-enhancing water. Our finding that Koisio media did not affect the mitochondrial membrane potential has suggested that novel mechanisms may underlie the effects of Koisio media on cellular energy.


title: A Morphogenetic Wave that Generates Mesenchymal-to-Epithelial Transition in the Lateral Plate Mesoderm

abstract:
Most mesodermal cells undergo multiple cycles of transition between an epithelial and mesenchymal state during embryonic development. While many studies have addressed the process of epithelial-to-mesenchymal transition (EMT), comparatively less is known regarding the complementary process, mesenchymal-to-epithelial transition (MET), which is essential for organogenesis and has also been proposed to be important for cancer metastasis. The current study investigated MET using the lateral plate mesoderm (LPM) of the chick embryo as a model system. We find that MET in the LPM proceeds as a wave, which divides the LPM into distinct mesenchymal, transition, and epithelial zones. In the multilayered mesenchymal zone, many apical epithelial markers, including N-Cadherin (N-Cad), Par-3 and Zo-1, but not atypical protein kinase C (aPKC), are detected as dispersed, partially co-localizing aggregates associated with cell-cell contacts. The transition zone is characterized by the appearance of aPKC and the formation of rosette-like structures characterized by wedge-shaped cells that are apical-basal polarized, with strong co-localization of apical polarity markers, but not yet arranged into distinct epithelial sheets. The transition zone is also enriched in mitotic cells. Subsequently, the rosettes resolve into two well-defined epithelial sheets that constitute the coelomic epithelium, the lining of the internal body cavity.

Prior to any overt signs of apical-basal polarity, fibronectin (FN) begins to accumulate at the future basal side of the incipient epithelium. Interference with Extracellular Matrix (ECM)-integrin signaling through disruption of focal adhesion kinase (FAK) or Talin function hindered the normal progression of the epithelialization process. Cells with disrupted FAK or Talin function retained mesenchymal-like characteristics with respect to cellular morphology and apical-basal marker distribution.

We propose a two-stage process for MET in the LPM. Initially, in the polarization phase, ECM-integrin-dependent signaling imparts apical-basal polarity, culminating in the activation of aPKC, to drive cell intercalation and rosette formation. Subsequently in the resolution phase, polarized rosette cells, perhaps facilitated by the weakening of cell-cell interactions that occurs during mitosis, expand their apical surface, and spread out to form new connections laterally to their fully epithelial neighbors. This sequence of events is propagated as a wave through the LPM, thus generating an integrated coelomic epithelium.


title: Mutation patterns in SARS-COV-2 Alpha and Beta variants indicate non-neutral evolution

abstract:
Due to the emergence of new variants of the SARS-CoV-2 coronavirus, the question of how the viral genomes evolved, leading to the formation of highly infectious strains, becomes particularly important. Two early emergent strains, Alpha and Beta, characterized by a significant number of missense mutations, provide natural testing samples.

In this study we are exploring the history of each of the segregating sites present in Alpha and Beta variants of concern, to address the question whether defining mutations were accumulating gradually leading to the formation of sequence characteristic of these variants.

Our analysis exposes data features that suggest other than neutral evolution of SARS-CoV-2 genomes, leading to emergence of variants of concern. We observe only small number of possible combinations of mutations indicating rapid evolution of genomes. In addtion, mutation patterns observed in whole genome samples of Alpha and Beta variants also indicate presence of stronger selection than in remaining genome samples.


title: Greenscreen decreases Type I Errors and increases true peak detection in genomic datasets including ChIP-seq

abstract:
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is used widely to identify both factor binding to genomic DNA and chromatin modifications. Analysis of ChIP-seq data is impacted by regions of the genome which generate ultra-high artifactual signals. To remove these signals from ChIP-seq data, ENCODE developed blacklists, comprehensive sets of regions defined by low mappability and ultra-high signals for human, mouse, worm, and flies. Currently, blacklists are not available for many model and non-model species. Here we describe an alternative approach for removing false-positive peaks we called "greenscreen". Greenscreen is facile to implement, requires few input samples, and uses analysis tools frequently employed for ChIP-seq. We show that greenscreen removes artifact signal as effectively as blacklists in Arabidopsis and human ChIP-seq datasets while covering less of the genome, dramatically improving ChIP-seq data quality. Greenscreen filtering reveals true factor binding overlap and of occupancy changes in different genetic backgrounds or tissues. Because it is effective with as few as three inputs, greenscreen is readily adaptable for use in any species or genome build. Although developed for ChIP-seq, greenscreen also identifies artifact signals from other genomic datasets including CUT&RUN. Finally, we present an improved ChIP-seq pipeline which incorporates greenscreen, that detects more true peaks than published methods.

One Sentence SummaryA facile method for removing artifact signal from ChIP-seq that improves downstream analyses


title: An amyloidosis-associated polymorphism does not alter LECT2 stability in vitro

abstract: Amyloid fibrils formed from leukocyte chemotactic factor 2 (LECT2), a secreted human cytokine, are associated with kidney failure in the disease amyloid LECT2 (ALECT2) amyloidosis. This rare disease was recognized in 2008 and has a variable prevalence worldwide. The mechanisms which lead to ALECT2 fibril deposition are not known and there are no treatments other than kidney transplant. The LECT2 gene harbors a single nucleotide polymorphism that leads to either a valine or isoleucine residue at position 40 of the mature protein. Most of the individuals diagnosed with ALECT2 amyloidosis are homozygous for valine at this position, which led us to hypothesize that the valine-containing variant of LECT2 protein is less stable and more prone to aggregation than the isoleucine-containing variant. Here, we investigate the structure, stability and aggregation of both variants of recombinant LECT2. Both variants have similar structures in solution; unfold in similar concentrations of urea; and aggregate at similar rates under native-like conditions, forming structures that bind to thioflavin T. Chelation of the structural zinc ion destabilizes both variants to a similar extent, and increases the rate at which they aggregate. We do not observe a consistent difference in stability or aggregation between the variants of LECT2, so we suggest that the presence of the valine residue at position 40 does not determine whether an individual is at increased risk of ALECT2 amyloidosis.