Transcriptional regulation of HSCs in Aging and MDS reveals DDIT3 as a Potential Driver of Dyserythropoiesis Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in elderly individuals. In this work, we analyzed the transcriptome of human HSCs purified from young and elderly healthy donors, as well as MDS patients, identifying transcriptional alterations following eight different patterns of expression. While aging-associated lesions seemed to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detected the upregulation of the transcription factor DDIT3. Overexpression of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis. Such effect was associated with downregulation of transcription factors required for normal erythropoiesis, and with a failure in the activation of their transcriptional programs. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia was able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing MDS patients.

STATEMENT OF SIGNIFICANCEThis study defines how human aging and MDS development are characterized by transcriptional alterations in HSCs that follow different patterns, some of which may contribute to myeloid transformation. Among them, we demonstrate how MDS-specific upregulation of DDIT3 in HSCs induces dyserythropoiesis, while its knockdown in HSPCs from MDS patients restores proper erythroid differentiation.
FGFR2 fusion protein-driven mouse models of intrahepatic cholangiocarcinoma unveil a necessary role for Erk signaling Background and aimsAbout 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutationally inactivated TP53, CDKN2A or BAP1. FFs span residues 1-768 of FGFR2 fused to sequences encoded by any of a long list (>60) of partner genes, a configuration sufficient to ignite oncogenic FF activation. In line, FGFR-specific tyrosine kinase inhibitors (F-TKI) were shown to provide clinical benefit in FF+ iCCA, although responses were partial and/or limited by resistance mechanisms, including the FF V565F gatekeeper mutation. Herein we present an FF-driven murine iCCA model and exploit its potential for pre-clinical studies on FF therapeutic targeting.

MethodsFour iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by intrahepatic/subcutaneous transplantation in immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies.

ResultsTumors diagnosed as CCA were obtained upon transplantation of FF-expressing liver organoids. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or presence of V565F mutation. Double blockade of FF-Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo.

ConclusionsFF-driven iCCA pathogenesis was successfully modelled in murine Tp53-/- background. This model revealed biological heterogeneity among structurally different FFs. Double blockade of FF-Erk signaling deserves consideration for improving precision-based approaches against human FF+ iCCA.

Abbreviations used in this paper: ANOVA, analysis of variance; Bap1, BRCA1-Associated-Protein 1; Cdkn2a, cyclin-dependent kinase inhibitor 2A; Cftr, cystic fibrosis transmembrane conductance regulator; Ck19, cytokeratin 19; Cyp3A, cytochrome P450, family 3, subfamily A; EGF, Epidermal growth factor; EGFR, Epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; Erk, extracellular signal-regulated kinase; FGFR2, fibroblast growth factor receptor 2; FRS2, fibroblast growth factor receptor substrate 2; GRB2, growth factor receptor-bound 2; GSEA, gene set enrichment analysis; GSVA, gene set variation analysis; H&E, hematoxylin and eosin; HepPar1, hepatocyte Paraffin 1; Hnf4-7, hepatocyte nuclear factor 4 alpha; Hprt, hypoxanthine-guanine phosphoribosyl transferase; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; NTRK, neurotrophic Tyrosine Kinase; Parp, Poly (ADP-ribose) polymerase; RECIST, response evaluation criteria in solid tumors; SHP2, Src homology phosphatase 2; Ttr, transthyretin.
Nanocrown electrodes for reliable and robust intracellular recording of cardiomyocytes and cardiotoxicity screening Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. However, the conventional patch clamp techniques for measuring iAPs are either laborious and low throughput or not suitable for measuring electrically connected cardiomyocytes. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. Nanoelectrodes show great promise, but the large variability in success rate, signal strength, and the low throughput of device fabrication have hindered them from being broadly adopted for proarrhythmia drug assessment. In this work, we developed vertically-aligned and semi-hollow nanocrown electrodes that are mechanically robust and made through a scalable fabrication process. Nanocrown electrodes achieve >99% success rates in obtaining intracellular access through electroporation, allowing reliable and simultaneous iAP recordings from up to 57 human pluripotent stem-cell-derived cardiomyocytes (hPSC-CMs). The accuracy of nanocrown electrode recordings is validated by simultaneous patch clamp recording from the same cell. Nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four to five incremental drug doses. In this way, the dose-response data is self-referencing, which avoids the cell-to-cell variations inherent to hPSC-CMs. We are hopeful that this technology development is a step towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity. Brominated Lipid Probes Expose Structural Asymmetries in Constricted Membranes Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane remodeling system, we observed leaflet-level and protein-localized lipid structural patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates, and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated SDPC showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl (DHA) tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

One-Sentence SummaryCryo-EM imaging of halogenated lipids and MD simulations provide molecular-scale insight into constricted bilayer structure. Unique, shared, and dominant brain activation in Visual Word Form Area and Lateral Occipital Complex during reading and picture naming Identifying printed words and pictures concurrently is ubiquitous in daily tasks, and so it is important to consider the extent to which reading words and naming pictures may share a cognitive-neurophysiological functional architecture. Two functional magnetic resonance imaging (fMRI) experiments examined whether reading along the left ventral occipitotemporal region (vOT; often referred to as a visual word form area, VWFA) has activation that is overlapping with referent pictures (i.e., both conditions significant and shared, or with one significantly more dominant) or unique (i.e., one condition significant, the other not), and whether picture naming along the right lateral occipital complex (LOC) has overlapping or unique activation relative to referent words. Experiment 1 used familiar regular and exception words (to force lexical reading) and their corresponding pictures in separate naming blocks, and showed dominant activation for pictures in the LOC, and shared activation in the VWFA for exception words and their corresponding pictures (regular words did not elicit significant VWFA activation). Experiment 2 controlled for visual complexity by superimposing the words and pictures and instructing participants to either name the word or the picture, and showed primarily shared activation in the VWFA and LOC regions for both word reading and picture naming, with some dominant activation for pictures in the LOC. Overall, these results highlight the importance of including exception words to force lexical reading when comparing to picture naming, and the significant shared activation in VWFA and LOC serves to challenge specialized models of reading or picture naming. Teosinte mexicana introgression modulates phosphatidylcholine levels and maize flowering time Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp.parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability, and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern US, Canada and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs. Impact of circadian time of dosing on cardiomyocyte-autonomous effects of glucocorticoids Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic oncedaily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing. In WT mice, we found that prednisone improved cardiac content of NAD+ and ATP with light-phase dosing (ZT0), while the effects were blocked by dark-phase dosing (ZT12). The drug effects on mitochondrial function were cardiomyocyte-autonomous, as shown by inducible cardiomyocyte-restricted glucocorticoid receptor (GR) ablation, and depended on an intact cardiomyocyte clock, as shown by inducible cardiomyocyte-restricted ablation of Brain and Muscle ARNT-like 1 (BMAL1). Conjugating time-of-dosing with chronic intermittence, we found that once-weekly prednisone improved metabolism and function in heart after myocardial injury dependent on circadian time of intake, i.e. with lightphase but not dark-phase dosing. Our study identifies cardiac-autonomous mechanisms through which circadian-specific intermittent dosing reconverts glucocorticoid drugs to metabolic boosters for the heart. 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">
View larger version (34K):
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. Liberation from fibrogenesis or tumorigenesis via cellular senescence by a novel small molecule BackgroundUncontrolled proliferative diseases such as fibrosis or cancer can be fatal. We previously found that a compound containing a chromone scaffold, ONG41008, had potent anti-fibrotic effects in diseased human lung myofibroblasts but not normal human lung fibroblasts.

MethodsWe investigated the effects of ONG41008 on tumor cells, and compared these effects with those in pathogenic myofibrotic cells and normal fibroblasts cells.

FindingsStimulation of A549 lung carcinoma epithelial cells with ONG41008 resulted in cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. Replicative senescence of A549 cells resulted in multinucleation, which was followed by oncogene-induced senescence. There was significant upregulation of expression and nuclear translocation of p-TP53 and p16 in ONG41008-treated A549 cells, and all cells died after 72 hr. Similar effects occurred after ONG41008 treatment in several human aggressive cancer cell lines such as PANC1, MCF7, PC3, or primary non-small cell lung carcinoma cells. Unlike cisplatin, ONG41008 was not toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio or increased lactate dehydrogenase levels in aged or diseased cells.

InterpretationTo our best knowledge, this is the first study to show that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis in vitro. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSThe notion that aging is a disease and that diseases occur as a consequence of aging was first put forward by David Sinclair and colleagues (Aging Cell;14(4):497-510). There are biological systems that provide evidence for this notion; for example, somatic cells can revert to embryonic cells, producing younger somatic cells. This phenomenon underlies induced pluripotent stem cells. Another example is that some types of jellyfish can live forever. These examples suggest that a counter-aging program exists in animals. Human diseases are the manifestations of cell aging generated by the accumulation of somatic mutations. Aged and pathogenic cells are senescent, so a drug that specifically targeted senescent cells might initiate a cellular program that could ameliorate age-associated disease. Indeed, the kinase inhibitor dasatinib induces cellular senescence (Clin Ther 2007 29:2289-2308). In 2017, two drugs that target senescent cells were identified: quercetin and fisetin. These drugs selectively kill senescent cells, and are referred to as senolytic drugs or senotherapeutics (Aging 2017 8;9(3):955-963). Although it is established that senescent cells accumulate in cancer and idiopathic pulmonary fibrosis (Nat Commun 2017 23;8 :14532), the effect of senolytic drugs in these diseases is largely unknown.

Added value of this studyThis study characterized a novel drug, termed ONG41008, which was found to have both senogenic and senolytic effects in cell-based assays. ONG41008 induced senescence in myofibroblasts and several cancer cell lines representative of aggressive human cancers, which was followed by cell death. Importantly, ONG41008 exhibited essentially no toxicity on normal human lung fibroblasts or primary prostate epithelial cells.

Implication of all the evidenceBased on our results, we believe that ONG41008 is a potent inducer of cellular senescence (replicative senescence and oncogene-induced senescence) and causes arrest of uncontrolled, pathogenic proliferation of myofibroblasts or cancer cells. AgMESH, a peritrophic matrix-associated protein embedded in Anopheles gambiaemelanotic capsules modulates malaria parasite infection Melanins are structurally complex pigments produced by organisms in all domains of life. In insects, melanins are essential for survival and have key roles in cuticle sclerotization, wound healing and innate immunity. In this study, we used a diverse set of molecular, biochemical, and imaging approaches to characterize mosquito melanin involved in innate immune defense (melanotic capsules). We observed that melanotic capsules enclosing Plasmodium berghei ookinetes were composed of an acid-resistant and highly hydrophobic material with granular appearance, which are characteristic properties of melanins. Spectroscopical analyses reveal chemical signatures of eumelanins and pheomelanin. Furthermore, we identified a set of 14 acid-resistant mosquito proteins embedded within the melanin matrix possibly related to an anti-Plasmodium response. Among these, AgMESH, a mucin-related protein highly conserved among insects that is associated with the midgut brush border microvilli proteome of Anopheles gambiae and A. albimanus. AgMESH gene silencing in mosquitos was associated with reduced Plasmodium parasite infection, compromised integrity of the peritrophic matrix, and inability to synthesize a dityrosine network. Our results provide a new approach to study aspects of insect melanogenesis that revealed proteins associated with melanotic capsule, one of which was strongly implicated in the stabilization of the peritrophic matrix and pathogenesis of Plasmodium spp. mosquito infection. Given the conservation of AgMESH among disease-transmitting insect vector species, future analysis of this protein could provide fertile ground for the identification of strategies that block transmission of vector borne diseases to humans.

Significance StatementMalaria is a parasitic disease transmitted by mosquito bites. Here, we adapt methodologies to study fungal melanogenesis to explore the melanin-based immune response of Anopheles gambiae against malaria parasites. We reveal that melanotic capsules against Plasmodium are composed of pheomelanin and eumelanin. We demonstrate that melanin-encapsulated Plasmodium is associated to acid-resistant mosquito gut proteins and identify several putative factors of the melanin-mediated immunity. Disruption of AgMESH, a surface-associated protein conserved among other mosquito vectors, demonstrates its ability to impaired formation of the dityrosine network and peritrophic matrix compromising parasite development within the mosquito gut. Our study provides a new approach to investigate the melanin-based defense mechanism in insects and identified a potential host molecule for developing novel universal vector-control schemes. TRIM28-dependent SUMOylation protects the adult ovary from the testicular pathway Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway. An Essential Role of UBXN3B in B Lymphopoiesis Hematopoiesis is finely regulated to enable timely production of the right numbers and types of mature immune cells to maintain tissue homeostasis. Dysregulated hematopoiesis may compromise antiviral immunity and/or exacerbate immunopathogenesis. Herein, we report an essential role of UBXN3B in maintenance of hematopoietic homeostasis and restriction of immunopathogenesis during respiratory viral infection. Ubxn3b deficient (Ubxn3b-/-) mice are highly vulnerable to SARS-CoV-2 and influenza A infection, characterized by more severe lung immunopathology, lower virus-specific IgG, significantly fewer B cells, but more myeloid cells than Ubxn3b+/+ littermates. This aberrant immune compartmentalization is recapitulated in uninfected Ubxn3b-/- mice. Mechanistically, UBXN3B controls precursor B-I (pre-BI) transition to pre-BII and subsequent proliferation in a cell-intrinsic manner, by maintaining BLNK protein stability and pre-BCR signaling. These results reveal an essential role of UBXN3B for the early stage of B cell development. Inter-tissue convergence of gene expression during ageing suggests age-related loss of tissue and cellular identity Developmental trajectories of gene expression may reverse in their direction during ageing, a phenomenon previously linked to cellular identity loss. Our analysis of cerebral cortex, lung, liver and muscle transcriptomes of 16 mice, covering development and ageing intervals, revealed widespread but tissue-specific ageing-associated expression reversals. Cumulatively, these reversals create a unique phenomenon: mammalian tissue transcriptomes diverge from each other during postnatal development, but during ageing, they tend to converge towards similar expression levels, a process we term Divergence followed by Convergence, or DiCo. We found that DiCo was most prevalent among tissue-specific genes and associated with loss of tissue identity, which is confirmed using data from independent mouse and human datasets. Further, using publicly available single-cell transcriptome data, we showed that DiCo could be driven both by alterations in tissue cell type composition and also by cell-autonomous expression changes within particular cell types. Assessing the impact of grassland management on landscape multifunctionality Land-use intensification has contrasting effects on different ecosystem services, often leading to land-use conflicts. While multiple studies have demonstrated how landscape-scale strategies can minimise the trade-off between agricultural production and biodiversity conservation, little is known about which land-use strategies maximise the landscape-level supply of multiple ecosystem services (landscape multifunctionality), a common goal of stakeholder communities.

We combine comprehensive data collected from 150 German grassland sites with a simulation approach to identify landscape compositions, with differing proportions of low-, medium-, and high-intensity grasslands, that minimise trade-offs between the six main grassland ecosystem services prioritised by local stakeholders: biodiversity conservation, aesthetic value, productivity, carbon storage, foraging, and regional identity. Results are made accessible through an online tool that provides information on which compositions best meet any combination of user-defined priorities (https://neyret.shinyapps.io/landscape_composition_for_multifunctionality/).

Results show that an optimal landscape composition can be identified for any pattern of ecosystem service priorities. However, multifunctionality was similar and low for all landscape compositions in cases where there are strong trade-offs between services (e.g. aesthetic value and fodder production), where many services were prioritised, and where drivers other than land use played an important role. We also found that if moderate service levels are deemed acceptable, then strategies in which both high and low intensity grasslands are present can deliver landscape multifunctionality. The tool presented can aid informed decision-making by predicting the impact of future changes in landscape composition, and by allowing for the relative roles of stakeholder priorities and biophysical trade-offs to be understood by scientists and practitioners alike.

HighlightsO_LIAn online tool identifies optimal landscape compositions for desired ecosystem services
C_LIO_LIWhen the desired services are synergic, the optimum is their common best landscape composition
C_LIO_LIWhen the desired services trade-off, a mix of grassland intensity is most multifunctional
C_LIO_LISuch tools could support decision-making processes and aid conflict resolution
C_LI

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/208199v5_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@3273f6org.highwire.dtl.DTLVardef@5b3a5aorg.highwire.dtl.DTLVardef@153f526org.highwire.dtl.DTLVardef@103cbea_HPS_FORMAT_FIGEXP  M_FIG C_FIG Mitochondrial double-stranded RNAs as a pivotal mediator in the pathogenesis of Sjogrens syndrome ObjectiveSjogrens syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. This study investigates the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFN response in SS.

MethodsSaliva and tear from SS patients and controls (n=73 for saliva and n=16 for tear), the salivary glands of the SS-prone-non-obese-diabetic mouse, and primary human salivary glandular cells were screened for mt-dsRNAs by RT-qPCR. The human salivary cell line (NS-SV-AC) grown as three-dimensional spheroids were subject to dsRNA stress to measure mt-dsRNA induction and recapitulation of SS glandular inflammatory features. Acetylcholine, SS-IgG, upadacitinib (JAK1 inhibitor), or 2-C'-methyladenosine (mitochondrial transcription inhibitor) were applied to characterize the roles of mt-dsRNAs. To identify endogenous dsRNA-sensor and confirm the mitochondrial origin of cytoplasmic dsRNAs, the immunoprecipitation of dsRNAs was performed.

Resultsmt-dsRNAs were elevated in the SS specimens with salivary ND5 and tear CYTB1 being statistically associated with secretory dysfunction/inflammation and corneal/conjunctival damage, respectively. Stimulation of the spheroids with dsRNA stress of poly I:C induced mt-dsRNAs, p-PKR, and I-IFNS via the JAK1/STAT pathway whereas the inhibition of mt-RNA synthesis or JAK1 attenuated the glandular signature. The inhibitory effect of acetylcholine on mt-dsRNAs and I-IFNS induction was reversed by SS-IgG.

Conclusionmt-dsRNAs amplify the impact of dsRNA stress on SS glandular signatures in vitro, potentially propagating a pseudo-viral signal in the SS target tissue.

SummaryMitochondrial double-stranded RNA levels were elevated in the tear and saliva of SS patients, which was associated with secretory dysfunction and tissue inflammation. These RNAs amplified type I interferon signature as well as glandular phenotypes reported in SS. Inhibitors of mitochondrial RNA transcription or JAK1 in salivary gland acinar cell spheroids attenuated the mitochondrial RNA-mediated changes. Protein contact map prediction using multiple sequence alignment dropout and consistency learning for sequences with fewer homologs The prediction of protein contact map needs enough normalized number of effective sequence (Nf) in multiple sequence alignment (MSA). When Nf is small, the predicted contact maps are often not satisfactory. To solve this problem, we randomly selected a small part of sequence homologs for proteins with large Nf to generate MSAs with small Nf. From these MSAs, input features were generated and were passed through a consistency learning network, aiming to get the same results when using the features generated from the MSA with large Nf. The results showed that this method effectively improves the prediction accuracy of protein contact maps with small Nf. Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible. 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. An estimate of the deepest branches of the tree of life from ancient vertically-evolving genes Core gene phylogenies provide a window into early evolution, but different gene sets and analytical methods have yielded substantially different views of the tree of life. Trees inferred from a small set of universal core genes have typically supported a long branch separating the archaeal and bacterial domains. By contrast, recent analyses of a broader set of non-ribosomal genes have suggested that Archaea may be less divergent from Bacteria, and that estimates of inter-domain distance are inflated due to accelerated evolution of ribosomal proteins along the inter-domain branch. Resolving this debate is key to determining the diversity of the archaeal and bacterial domains, the shape of the tree of life, and our understanding of the early course of cellular evolution. Here, we investigate the evolutionary history of the marker genes key to the debate. We show that estimates of a reduced Archaea-Bacteria (AB) branch length result from inter-domain gene transfers and hidden paralogy in the expanded marker gene set. By contrast, analysis of a broad range of manually curated marker gene datasets from an evenly sampled set of 700 Archaea and Bacteria reveal that current methods likely underestimate the AB branch length due to substitutional saturation and poor model fit; that the best-performing phylogenetic markers tend to support longer inter-domain branch lengths; and that the AB branch lengths of ribosomal and non-ribosomal marker genes are statistically indistinguishable. Furthermore, our phylogeny inferred from the 27 highest-ranked marker genes recovers a clade of DPANN at the base of the Archaea, and places CPR within Bacteria as the sister group to the Chloroflexota. Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. Multivariate genome-wide association study on tissue-sensitive diffusion metrics identifies key molecular pathways for axonal growth, synaptogenesis, and astrocyte-mediated neuroinflammation The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discovered 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% were validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development were found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders. Deficits in seizure threshold and other behaviors in adult mice without gross neuroanatomic injury after late gestation transient prenatal hypoxia Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain. High performance single-cell gene regulatory network inference at scale: The Inferelator 3.0 MotivationGene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. Methods for inferring and reconstructing networks from genomics data have evolved rapidly over the last decade in response to advances in sequencing technology and machine learning. The scale of data collection has increased dramatically; the largest genome-wide gene expression datasets have grown from thousands of measurements to millions of single cells, and new technologies are on the horizon to increase to tens of millions of cells and above.

ResultsIn this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator is able to integrate the largest single-cell datasets and learn cell-type specific gene regulatory networks. Compared to other network inference methods, the Inferelator learns new and informative Saccharomyces cerevisiae networks from single-cell gene expression data, measured by recovery of a known gold standard. We demonstrate its scaling capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression dataset with paired single-cell chromatin accessibility data.

AvailabilityThe inferelator software is available on GitHub (https://github.com/flatironinstitute/inferelator) under the MIT license and has been released as python packages with associated documentation (https://inferelator.readthedocs.io/). Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important.