What is valued in conservation? A framework to compare ethical perspectives Perspectives in conservation are based on a variety of value systems. Such differences in how people value nature and its components lead to different evaluations of the morality of conservation goals and approaches, and often underlie disagreements in the formulation and implementation of environmental management policies. Specifically, whether a conservation action (e.g. killing feral cats to reduce predation on bird species threatened with extinction) is viewed as appropriate or not can vary among people with different value systems. Here, we present a conceptual, mathematical framework intended as a tool to systematically explore and clarify core value statements in conservation approaches. Its purpose is to highlight how fundamental differences between these value systems can lead to different prioritizations of available management options and offer a common ground for discourse. The proposed equations decompose the question underlying many controversies around management decisions in conservation: what or who is valued, how, and to what extent? We compare how management decisions would likely be viewed under three different idealised value systems: ecocentric conservation, which aims to preserve biodiversity; new conservation, which considers that biodiversity can only be preserved if it benefits humans; and sentientist conservation, which aims at minimising suffering for sentient beings. We illustrate the utility of the framework by applying it to case studies involving invasive alien species, rewilding, and trophy hunting. By making value systems and their consequences in practice explicit, the framework facilitates debates on contested conservation issues, and complements philosophical discursive approaches about moral reasoning. We believe dissecting the core value statements on which conservation decisions are based will provide an additional tool to understand and address conservation conflicts.
Limited selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.
A platform of patient-derived microtumors identifies individual treatment responses and therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment. Protein cost minimization promotes the emergence of coenzyme redundancy Coenzymes distribute a variety of chemical moieties throughout cellular metabolism, participating in group (e.g., phosphate, acyl) and electron transfer. For a variety of reactions requiring acceptors or donors of specific resources, there often exist degenerate sets of molecules (e.g., NAD(H) and NADP(H)) that carry out similar functions. Although the physiological roles of various coenzyme systems are well established, it is unclear what selective pressures may have driven the emergence of coenzyme redundancy. Here we use genome-wide metabolic modeling approaches to decompose the selective pressures driving enzymatic specificity for either NAD(H) or NADP(H) in the metabolic network of Escherichia coli. We found that few enzymes are thermodynamically constrained to using a single coenzyme, and in principle, a metabolic network relying on only NAD(H) is feasible. However, structural and sequence analyses revealed widespread conservation of residues that retain selectivity for either NAD(H) or NADP(H), suggesting that additional forces may shape specificity. Using a model accounting for the cost of oxidoreductase enzyme expression, we found that coenzyme redundancy universally reduces the minimal amount of protein required to catalyze coenzyme-coupled reactions, inducing individual reactions to strongly prefer one coenzyme over another when reactions are near thermodynamic equilibrium. We propose that protein minimization generically promotes coenzyme redundancy, and that coenzymes typically thought to exist in a single pool (e.g., CoA) may exist in more than one form (e.g., dephospho-CoA).

Significance statementMetabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes (e.g., NAD(H) and NADP(H)) associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling, and structural and sequence analysis indicates that coenzyme redundancy is not essential for metabolism, but rather an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes, and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. 2,3-cAMP treatment mimics stress molecular response in Arabidopsis thaliana The role of the RNA degradation product 2,3-cyclic adenosine monophosphate (2,3-cAMP) is poorly understood. Recent studies have identified 2,3-cAMP in plant material and determined its role in stress signaling. The level of 2,3-cAMP increases upon wounding, dark, and heat, and 2,3-cAMP by binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insight into 2,3-cAMP function, we used a multi-omics approach combining transcriptomics, metabolomics, and proteomics to dissect Arabidopsis response to 2,3-cAMP treatment. We demonstrated that 2,3-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway from human cells might also exist in plants. Transcriptomic analysis revealed only minor overlap between 2,3-cAMP-and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2,3-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses including protein and RNA degradation products, glucosinolates, chaperones and SG components. Finally, we demonstrated that 2,3-cAMP treatment influences the movement of processing bodies, supporting the role of 2,3-cAMP in the formation and motility of membraneless organelles. Mobile element integration reveals a chromosome dimer resolution system in Legionellales. In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The concatenated DNA must be resolved before cell separation to ensure genomic stability and cell viability. The broadly conserved dif/Xer system counteracts the formation of dimers by catalyzing one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site-mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (herein named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site, and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Lastly, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids.

Significance StatementThe maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers as they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif, leading to rapid site-specific recombination and elimination of the duplicated intervening sequence. The apparent absence of chromosome-dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens. Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability BackgroundVariability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae, and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range.

ResultsWe show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms.

ConclusionsThis study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in Zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way. Strong immobilization of C. elegans en masse on cultivation plates by thermoelectric cooling for high-throughput submicron-resolution microscopy Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows. Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies Kaposis sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposis sarcoma (KS). We previously demonstrated that expression of the KSHV protein, Kaposin B (KapB), promoted inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). Processing bodies (PBs) modify gene expression by silencing or degrading labile messenger RNAs (mRNAs) including many transcripts that encode inflammatory or angiogenic proteins that are associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via the phosphorylation of the autophagy regulatory protein, Beclin 1. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin 1 phosphorylation or autophagic flux. Moreover, we reveal that PB disassembly mediated by KSHV or KapB depended on canonical autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of the inflammatory, endothelial cell cancer, Kaposis Sarcoma (KS). KSHV induces a pro-tumourigenic inflammatory environment which aids in the establishment and maintenance of the KS lesion. Processing bodies (PBs) are cellular structures that dampen inflammatory gene expression by suppression or decay of their cognate RNA molecules. We previously showed that the viral protein KapB caused PB disappearance during KSHV infection, identifying a new pathway used by KSHV to elicit inflammation. Now, we show that KSHV and KapB hijack the cellular degradative process of autophagy to promote PB disassembly and increase inflammatory gene expression. This places autophagy as central to the regulation of inflammation by KSHV and illustrates another remarkable strategy used by these viruses to create a tumourigenic microenvironment. Immune complex solubility and size govern individual Fc-gamma receptor responses: Novel biomarkers for autoimmune diseases Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold. Alphaviruses such as Ross River virus (RRV), chikungunya virus, Sindbis virus (SINV), and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 and 1.9 [A]. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogendeuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Key PointsO_LICrystal structures of alphavirus nsP4 RNA polymerase domain from RRV and SINV.
C_LIO_LInsP4 protein is highly dynamic with an intrinsically disordered N-terminal domain.
C_LIO_LIOptimized RNA elongation activity assay to facilitate antiviral discovery.
C_LI Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Frequency modulated timer regulates mammalian hibernation Mammalian hibernators decrease basal metabolism and body temperature (Tb) to minimize energy expenditure in harsh seasons. During hibernation, Tb drops to low temperature (<10 {degrees}C) and remains constant for days, known as deep torpor in small mammalian hibernators. Spontaneous interbout arousals interrupt torpor bouts, when Tb recovers to euthermic state ~37 {degrees}C. Torpor-interbout arousal event repeats during several months of hibernation. However, little is known about mechanisms governing Tb fluctuation across torpor-interbout arousal cycles during hibernation. Recent improvement in data-logging techniques enables us to monitor Tb for more than hundred days with high precision, opening up new avenues for quantitative analysis to address the principle governing Tb fluctuation. Here, we analyzed Tb fluctuation across torpor-interbout arousal cycle of Syrian hamster, which can hibernate in response to chronic cold and short photoperiod under a laboratory condition, using generalized harmonic analysis and discovered a model with frequency modulation quantitatively reproducing Tb fluctuation. This analysis also identified an unexpectedly longer period of 120-430 days as the period that modulates another period of several days, generating Tb fluctuation for Syrian hamster. We propose that concerted action of two endogenous periods governs torpor-interbout arousal cycles during hibernation. EEG-based neurofeedback with network components extraction: a data-driven approach by multilayer ICA extension and simultaneous EEG-fMRI measurements Several studies have reported advanced treatments for depressive symptoms, such as real-time neurofeedback (NF) with functional MRI (fMRI) and/or electroencephalogram (EEG). NF focusing on a regularization of brain activity associated with the amygdala or functional connectivity (FC) between the executive control network (ECN) and default mode network (DMN) has been applied to reduce depressive symptoms. However, it is practically difficult to install the fMRI-NF system and to consistently provide treatment, because of high cost. Additionally, no practical signal processing techniques have been developed extracting FC-related features from EEG signals, particularly when no physical forward models are available. In this regard, stacked pooling and linear components estimation (SPLICE), recently proposed as a multilayer extension of independent component analysis (ICA) and related independent subspace analysis (ISA), can be a promising alternative. The resting-state EEG network features can be correlated with fMRI network activity corresponding to the DMN or ECN. This may enable the modulation of the target FC-related features in EEG-based NF.

In this study, we developed a real-time EEG NF system for improving depressive symptoms by using the SPLICE. Utilizing information from the fMRI biomarkers, we evaluated our paradigm for effectiveness with regard to upregulation of the dorsolateral prefrontal cortex /middle frontal gyrus or downregulation of the precuneus/posterior cingulate cortex. We conducted an NF experiment in participants with subclinical depression; the participants were divided into the NF group (n=8) and the sham group (n=9). We found a significant reduction and a large effect size in the rumination response scale (RRS) score (reflection) in the NF group, compared to the sham group.

However, we did not find a significant relationship between the training score and difference in symptoms. This suggests that increased controllability of the EEG signals did not directly reduce the RRS reflection score. This could be due to various reasons such as improper feature extraction, individual differences, and the targeted brain regions. In this paper, we also discuss the possible ways to modify our NF protocol including the design of the experiment, sample size, and online processing. We then discuss way to improve the NF training, based on our results. Somatic uniparental disomy mitigates the most damaging EFL1 allele combination in Shwachman-Diamond syndrome Shwachman-Diamond syndrome (SDS; OMIM: #260400) is caused by variants in SBDS (Shwachman-Bodian-Diamond syndrome gene), which encodes a protein that plays an important role in ribosome assembly. Recent reports suggest that recessive variants in EFL1 are also responsible for SDS. However, the precise genetic mechanism that leads to EFL1-induced SDS remains incompletely understood. Here we present three unrelated Korean SDS patients that carry biallelic pathogenic variants in EFL1 with biased allele frequencies, resulting from a bone marrow-specific somatic uniparental disomy (UPD) in chromosome 15. The recombination events generated cells that were homozygous for the relatively milder variant, allowing for the evasion of catastrophic physiological consequences. Still, the milder EFL1 variant was solely able to impair 80S ribosome assembly and induce SDS features in cell line, zebrafish, and mouse models. The loss of EFL1 resulted in a pronounced inhibition of terminal oligo-pyrimidine element-containing ribosomal protein transcript 80S assembly. Therefore, we propose a more accurate pathogenesis mechanism of EFL1 dysfunction that eventually leads to aberrant translational control and ribosomopathy. The neurogenic fate of the hindbrain boundaries relies on Notch-dependent asymmetric cell divisions The generation of cell diversity in the central nervous system occurs during embryogenesis and requires a precise balance between cell proliferation, commitment to specific fates, and further neuronal differentiation. The cellular and molecular mechanisms regulating this balance in the embryonic brain are still poorly understood. Here we study how the neurogenic capacity in the embryonic hindbrain is spatiotemporally allocated, and how the fate and the growth of the hindbrain boundary cells are regulated. By generating a CRISPR-based knock-in zebrafish transgenic line to specifically label the hindbrain boundaries, we unveiled that boundary cells undergo a functional transition to become neurogenic during hindbrain segmentation concurrently as they maintain the progenitor cell pool. Boundary cells engaged in neurogenesis coinciding with the onset of Notch signaling, which triggered their asymmetrical cell division. Our findings reveal that distinct neurogenic phases take place during hindbrain growth and suggest that boundary cells contribute to refine the final number, identity, and proportion of neurons in the brain.

SUMMARYHindbrain boundary cells undergo a functional transition to become neurogenic concurrently as they are maintained as a progenitor cell pool. This involves a behavioral switch from symmetrically dividing progenitor cells to asymmetrically dividing progenitors, which depends on Notch-activity. Detecting phylodiversity-dependent diviersification with a general phylogenetic inference framework AO_SCPLOWBSTRACTC_SCPLOWDiversity-dependent diversification models have been extensively used to study the effect of ecological limits and feedback of community structure on species diversification processes, such as speciation and extinction. Current diversity-dependent diversification models characterise ecological limits by carrying capacities for species richness. Such ecological limits have been justified by niche filling arguments: as species diversity increases, the number of available niches for diversification decreases.

However, as species diversify they may diverge from one another phenotypically, which may open new niches for new species. Alternatively, this phenotypic divergence may not affect the species diversification process or even inhibit further diversification. Hence, it seems natural to explore the consequences of phylogenetic diversity-dependent (or phylodiversity-dependent) diversification. Current likelihood methods for estimating diversity-dependent diversification parameters cannot be used for this, as phylodiversity is continuously changing as time progresses and species form and become extinct.

Here, we present a new method based on Monte Carlo Expectation-Maximization (MCEM), designed to perform statistical inference on a general class of species diversification models and implemented in the R package emphasis. We use the method to fit phylodiversity-dependent diversification models to 14 phylogenies, and compare the results to the fit of a richness-dependent diversification model. We find that in a number of phylogenies, phylogenetic divergence indeed spurs speciation even though species richness reduces it. Not only do we thus shine a new light on diversity-dependent diversification, we also argue that our inference framework can handle a large class of diversification models for which currently no inference method exists. Analysis of Drosophila cardiac hypertrophy by micro-computerized tomography for genetic dissection of heart growth mechanisms Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

NEW AND NOTEWORTHYGenetic analysis of cardiac hypertrophy in Drosophila holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. However, this potential has been hindered by a lack of rapid and effective methods for analysis of heart size in flies. Here we demonstrate that analysis of the Drosophila heart with micro-computerized tomography yields accurate and highly reproducible heart size measurements that can be used to efficiently analyze heart growth and cardiac hypertrophy in Drosophila. Discovering Rare Cell Types through Information-based Dimensionality Reduction Dimensionality reduction is crucial to summarizing the complex transcriptomic landscape of single cell datasets for downstream analyses. However, current dimensionality reduction approaches favor large cellular populations defined by many genes, at the expense of smaller and more subtly-defined populations. Here, we present surprisal component analysis (SCA), a technique that leverages the information-theoretic notion of surprisal for dimensionality reduction, and demonstrate its ability to improve the representation of clinically important populations that are indistinguishable using existing pipelines. For example, in cytotoxic T-cell data, SCA cleanly separates the gamma-delta and MAIT cell subpopulations, which are not detectable via PCA, ICA, scVI, or a wide array of specialized rare cell recovery tools. We also show that, when used instead of PCA, SCA improves downstream imputation to more accurately restore mRNA dropouts and recover important gene-gene relationships. SCAs information-theoretic paradigm opens the door to more meaningful signal extraction, with broad applications to the study of complex biological tissues in health and disease. A novel nongenetic murine model of hyperglycemia and hyperlipidemia-associated aggravated atherosclerosis ObjectiveAtherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s).

Methods and ResultsWild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less -SMA-positive area) and had more inflammation (increased plasma IL-1{beta} level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks.

ConclusionWe established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. Mechanical coupling of supracellular stress amplification and tissue fluidization during exit from quiescence Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes and immunity. Here we show that quiescent human keratinocyte monolayers contain an actinomyosin-based system that facilitates global viscoelastic flow upon serum-stimulated exit from quiescence. Mechanistically, serum exposure causes rapid amplification of pre-existing contractile sites leading to a burst in monolayer stress that subsequently drives monolayer fluidization. The stress magnitude after quiescence exit correlates with quiescence depth, and a critical stress level must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility and identifies global stress amplification as a mechanism for tissue fluidization. A high throughput lipidomics method using scheduled multiple reaction monitoring Lipid compositions of cells, tissues and bio-fluids are complex, with varying concentrations and structural diversity, which makes their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based method for large-scale lipidomics using a combination of variable retention time window and relative dwell time weightage. Using this, we detected more than 1000 lipid species, including structural isomers. The limit of detection varied from femtomolar to nanomolar range and the coefficient of variance <30% for 849 lipid species. We used this method to identify lipids altered due to Vitamin B12 deficiency and found that the levels of lipids with {omega}-3 fatty acid chains decreased while those with {omega}-6 increased. This method enables identification of by far the largest number of lipid species with structural isomers in a single experiment and would significantly advance our understanding of the role of lipids in biological processes. High-quality genome assembly of Cinnamomum burmannii (chvar. Borneol) provides insights into the natural borneol biosynthesis Cinnamomum burmannii (chvar. Borneol) is a well-known medicinal and industrial plant cultivated in the Lingnan region of China. It is the key source from organism of natural borneol (D-borneol), one of the precious and widely used Chinese herbal medicines with a variety of medicinal effects. Here, we report a high-quality chromosome-scale genome assembly of C. burmannii (chvar. Borneol) using Pacbio single-molecule sequencing and Hi-C technology. The assembled genome size was 1.14 GB with a scaffold N50 of 94.30 Mb, while 98.77% of the assembled sequences were anchored on 12 pseudochromosomes including 41549 protein-coding genes. Genomic evolution analysis revealed C. burmannii and C. micranthum shared two Lauraceae unique ancestral whole-genome duplication (WGD) events. Likewise, comparative genomic analysis showed strong collinearity between these two species. Besides, the analysis for Long Terminal Repeat Retrotransposons (LTR-RTs) indicated the outbreak of LTR-RTs insertion made a great contribution to the size difference of genomes between C. burmannii and C. micranthum. Furthermore, the candidate genes in pathway associated with natural borneol synthesis were identified on the genome and their differential expressions were analyzed in various biological tissues. We considered that several of genes in Mevalonate (MVA) Methylerythritol Phosphate (MEP) pathways or in downstream pathway have the potential to be the key factors in the biosynthesis of D-borneol. We also constructed the genome database (CAMD; http://www.cinnamomumdatabase.com/) of Cinnamomum species for a better data utilization in the future. All these results will enrich the genomic data of Lauraceae plants and facilitate genetic improvement of this commercially important plant.