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.
A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis According to the current consensus, neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular-subventricular zone V-SVZ. Both processes will then ultimately lead to the generation of adult-born olfactory bulb (OB) interneurons. In contrast to this view, we here found that adult-born OB interneurons largely derive from an additional NSC type resident in the basal V-SVZ. Despite being both capable of self-renewal and long-term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch-related genes also differed between the two NSC groups and Notch-activation was greatest in apical NSCs. Apical downregulation of Notch-effector Hes1 decreased Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation-dependent increase in neurogenesis was paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis whereas apical NSCs impart Notch-mediated lateral inhibition across the V-SVZ.
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. HSP90 modulates T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis. 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. Onset and kinetics of B cell polarization depend on substrate mechanics and antigen concentration The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied.

Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how the ligand concentration, the surface fluidity and the substrate rigidity impact this polarization, we show that its onset depends on the antigen concentration at the synapse, and that the substrate rigidity controls both its onset and its kinetics. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Significance statementTo fulfill their immune function, B cells acquire antigens presented on the surface of antigen-presenting cells and organize their cytoskeleton and lysosomal traffic towards the contact. The polarization mechanism is evolutionary conserved, making the system interesting both from the immunology and the cell biology point of view. We developed and finely characterized antigen-coated droplets as surrogate presenting cells with a physiological-flow microfluidic cell-pairing chip to explore the B cell lysosome polarization in response to changes in droplet mechanics and antigen concentration. We observed that polarization is sensitive to mechanical properties (responding more strongly but less fast on stiffer substrates) and to the local antigen concentration at the contact. Hence, surprisingly, a late cell response is sensitive to the mechanics of the contact. Characterising ontogeny of quantity discrimination in zebrafish A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here we investigated the ontogeny of this ability using a group size preference task in juvenile zebrafish. Fish showed group size preference from 21 days post fertilization (dpf) and reliably chose the larger group when presented with discriminations of between 1 vs. 3, 2 vs. 5 and 2 vs. 3 conspecifics but not 2 vs. 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1:2, fish could discriminate between 1 vs. 2 individuals and 3 vs. 6, but again, not when given a choice between 2 vs. 4 individuals. These findings are in agreement with studies in other species suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fish may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasise the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition. BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic, however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/461749v2_ufig1.gif" ALT="Figure 1">
View larger version (53K):
org.highwire.dtl.DTLVardef@1e5e3c6org.highwire.dtl.DTLVardef@1cd0e60org.highwire.dtl.DTLVardef@16adf4corg.highwire.dtl.DTLVardef@16a0089_HPS_FORMAT_FIGEXP  M_FIG C_FIG Designing single-cell experiments to harvest fluctuation information while rejecting measurement noise Measurement error is a complicating factor that reduces or distorts the information contained in any experiment. This problem becomes even more serious in the context of experiments that are specifically designed to quantify single-cell gene expression heterogeneity, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. It is not clear how measurement noise should be managed, in addition to other experiment design variables such as sampling size and frequency, to ensure that collected data will provide useful insights on gene expression mechanisms of interest. To address these fundamental single-cell experiment design challenges, we propose a computational framework that makes explicit use of measurement error modeling and Fisher Information Matrix-based criteria to decide between experimental methods. This unified approach not only quantifies how different noise characteristics affect uncertainty in parameter estimation, but also enables a systematic approach to design hybrid experiments that combine different measurement modalities. First detection of herpesvirus and frequency of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Prenatal environmental conditions underlie alternative reproductive tactics that drive the formation of a mixed-kin cooperative society Although animal societies often evolve due to limited natal dispersal that results in kin clustering and facilitates cooperation among relatives, many species form cooperative groups with low and variable kin structure. Groups in such mixed-kin societies often comprise residents and immigrants of the same sex that compete for breeding opportunities. To understand how such mixed-kin societies form despite their potential for social conflict, we investigated the environmental causes and subsequent fitness consequences of dispersal decisions in male cooperatively breeding superb starlings (Lamprotornis superbus) that live in a climatically unpredictable environment. We show that the two alternative reproductive tactics--natal dispersal or philopatry--exhibit reproductive tradeoffs resulting in equal lifetime inclusive fitness. The tactic an individual adopts is governed by the environment its parents experience prior to laying rather than the environment it experiences during its juvenile stage. When individuals adopt the tactic not predicted by early life environmental conditions, their fitness is reduced. Ultimately, climate-driven oscillating selection may help stabilize mixed-kin animal societies, despite their reduced kin structure and potential for social conflict. Lipid membranes modulate the activity of RNA through sequence-dependent interactions RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here we show that lipid membranes can act as RNA organization platforms, introducing a novel mechanism for ribo-regulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and novel RNA-based lipid biosensors, and bear significant implications for RNA World scenarios for the origin of life. High-Speed, Cortex-Wide Volumetric Recording of Neuroactivity at Cellular Resolution using Light Beads Microscopy Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally-distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 108 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3x5x0.5 mm3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4x6x0.5 mm3 volumes at ~2Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain. High quality mapping of chromatin at or near the nuclear lamina for small numbers of cells The chromatin associated with the nuclear lamina (NL) is referred to as Lamina-Associated Domains (LADs). While mapping of this feature has been done using various technologies, technical limitations exist for each of the methods. Here, we present an adaptation of the Tyramide-Signal Amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50,000 cells without using carriers. The cTSA-seq mapped regions are composed of LADs and smaller chromatin regions that fall within the chromatin B-compartment known to be enriched for heterochromatin and be present at the nuclear periphery. As a proof of principle, we used cTSA-seq to map chromatin at or near the assembling NL as cells exit mitosis and progress through early and later G1. Consistent with previous reports, lamin-B1 based cTSA-seq revealed that regions toward the distal ends of chromosomes are near or at the reassembling NL during early G1. The cTSA-seq mapping and analyses revealed similarity between the early G1 chromatin and oncogene-induced senescent cell populations. The cTSA-seq reported here represents a useful method for analyzing chromatin at or near the NL from small numbers of cells. Modular biosynthesis of plant hemicellulose and its impact on yeast cells BackgroundThe carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past five years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes.

ResultsHere, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made {beta}-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana.

ConclusionThe results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as {beta}-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins. An environmentally responsive transcriptional state modulates cell identities during root development Roots are fundamental organs for plant development and response to their environment: they anchor the plant to its growth substrate, uptake nutrients and water vital to plant growth, and can sense and respond to a variety of biotic and abiotic stresses. The architecture of root systems and their growth are known to be strongly affected by the environmental conditions found in the soil. However, the acquisition of cell identities at the root meristem is still mainly viewed as ontogenetically driven, where a small number of stem cells generate all the cell types through stereotyped divisions followed by differentiation, along a simple developmental trajectory. The extent to which environmental cues precisely shape and affect these developmental trajectories remains an open question. We used single-cell RNA-seq, combined with spatial mapping, to deeply explore the trajectories of cell states at the tip of Arabidopsis roots, known to contain multiple developing lineages. Surprisingly, we found that most lineage trajectories exhibit a stereotyped bifid topology with two developmental trajectories rather than one. The formation of one of the trajectories is driven by a strong and specific activation of genes involved in the responses to various environmental stimuli, that affects only of a subset of the cells in multiple cell types simultaneously, revealing another layer of patterning of cell identities in the root that is independent of cell ontogeny. We demonstrate the robustness of this environmentally responsive transcriptional state by showing that it is present in a mutant where cell type identities are greatly perturbed, as well as in different Arabidopsis ecotypes. We also show that the root can adapt the proportion of cells that acquire this particular state in response to environmental signals such as nutrient availability. The discovery of this cell state reveals new layers of cell identity that may underpin the adaptive potential of plant development. A robust approach to estimate relative phytoplankton cell abundance from metagenomes Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Phytoplankton communities are generally studied by PCR amplification of bacterial (16S), nuclear (18S) or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. In addition, rRNA marker genes fail to capture both cyanobacteria and photosynthetic eukaryotes simultaneously. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the Tara Oceans datasets, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these organisms, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities. Avian haemosporidians (Plasmodium and Haemoproteus) status in selected bird groups (Old world Flycatchers, Warblers, Babblers, and Thrushes) of India and their phylogenetic relationships with other lineages of the world The avian haemosporidians (Plasmodium and Haemoproteus) are widely distributed and may affect the host populations from body damage at individual level to the extinction of a population. The knowledge about their status may help in future avifauna conservation plans. Hence, we examined the avian haemosporidians status, in selected bird groups (Old world Flycatchers, Warblers, Babblers, and Thrushes) of India, and their phylogenetic relationships with other known lineages of the world. We used the common genetic marker (Cytochrome b gene fragment of 479 bp) with information on the geographic distribution of parasite and host species available at MalAvi database. The prevalence of avian haemosporidians, from northern, eastern, and southern parts of India and phylogenetic genetic analysis of lineages was carried out to know the genetic relatedness among them at local and world level. The MCC tree revealed six Haemosporidian lineages in which one was common (H_MSP01) and five were unique (H_CYOPOL01, H_CHD01, H_CYORUB01, H_EUMTHA01, and P_GEOCIT01). The avian host richness Index was 2.0852. 9.9%, prevalence of Haemosporidian infection was found in 111 DNA samples belonging to 6 host species. The Haemoproteus prevalence was found to be 9.0 % across five host species (Phylloscopus trochiloides, Cyornis poliogenys, C. hainanus dialilaemus, C. rubeculoides, Eumiyas thalassinus) and Plasmodium prevalence was 0.9% in Geokichla citrina. Spatial phylogeny at global level showed H_MSP01 lineage, found in different host species in India, was genetically related to H. pallidus lineages (COLL2 and PFC1) in parts of Africa, Europe, North America, Malaysia, and Philippines. The Plasmodium lineage (P_GEOCIT01) was related to PADOM16 in Egypt with poor sequence similarity (93.89%). The statistical analysis suggested that the haemosporidians host species distribution range was directly and significantly associated with the altitude, minimum temperature, and relative humidity. H_MSP01 distribution was in accordance with H. pallidus having a wide geographic and host range. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ-9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Exploratory analysis of multiple traits co-adaptations in the population history During the history of range expansion, the populations encounter with variety of environments. They respond to the local environments by modifying the mutually interacting traits. Therefore, to understand the whole life history of the populations, it is ideal to capture the history of their range expansion with reference to the series of surrounding environments and to infer the coadaptation of the multiple traits. Toward this end, we provide an exploratory analysis based on the features of populations: site frequency spectra of populations, population-specific FST, association between genes and environments, positive selections on traits mapped on the admixture graph, and GWAS results. Correspondence analysis of genes, environments, and traits provides a birds-eye view of the history of population differentiation and range expansion and various types of environmental selections at the times. Principal component analysis of the estimated trait-specific polygenic adaptations mapped on the admixture graph enables to understand the coadaptation of multiple traits. The potential usefulness was confirmed by analyzing a public dataset of wild poplar in northwestern America. In response to the northern cold temperature and longer daylength, the populations increased the photosynthetic activity and nutrient use efficiency at the expense of the risk of pathogen invasion, and in response to warm temperature, they increased the growth. At higher altitude, they shifted the maximum activity to earlier period in spring to reduce the activity in dry summer. The R codes for our representation method and simulations of population colonization used in this study are available as supplementary script. Divergent Acyl Carrier Protein Decouples Mitochondrial Fe-S Cluster Biogenesis from Fatty Acid Synthesis in Malaria Parasites Plasmodium falciparum malaria parasites are early-diverging eukaryotes with many unusual metabolic adaptations. Understanding these adaptations will give insight into parasite evolution and unveil new parasite-specific drug targets. Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis. In yeast and humans, mACP also functions to biochemically couple FASII activity to electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast to most eukaryotes, the Plasmodium mitochondrion lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC followed by broader cellular defects. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of both Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other Apicomplexan pathogens, including Toxoplasma and Babesia. This discovery also highlights the ancient, fundamental role of ACP in mitochondrial Fe-S cluster biogenesis and unveils an evolutionary driving force to retain this interaction with ACP independent of its eponymous function in FASII.

Significance StatementPlasmodium malaria parasites are single-celled eukaryotes that evolved unusual metabolic adaptations. Parasites require a mitochondrion for blood-stage viability, but essential functions beyond the electron transport chain are sparsely understood. Unlike yeast and human cells, the Plasmodium mitochondrion lacks fatty acid synthesis enzymes but retains a divergent acyl carrier protein (mACP) incapable of tethering acyl groups. Nevertheless, mACP is essential for parasite viability by binding and stabilizing the core mitochondrial Fe-S cluster biogenesis complex via a divergent molecular interface lacking an acyl-pantetheine group that contrasts with other eukaryotes. This discovery unveils an essential metabolic adaptation in Plasmodium and other human parasites that decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis and evolved at or near the emergence of Apicomplexan parasitism. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. XRN2-mediated regulation of let-7 microRNAs is of critical pathophysiological significance in Humans MicroRNAs are critical regulators of diverse developmental and physiological processes, and they themselves get regulated both at the level of biogenesis and turnover. We demonstrate that the ribonuclease XRN2 can degrade the mature forms of certain let-7 family members in multiple human cancer cell lines, without affecting their precursors. XRN2 depletion results in a reduction in the expression of a number of oncogenes, and diminishes the proliferative and metastatic potential of cancer cells. The clinical relevance of these observations is also verified in tumour transcriptomics data from public RNA-sequencing datasets, where XRN2 mRNA expression is inversely correlated with the levels of a large number of miRNAs, including let-7 members, and high XRN2 mRNA levels are associated with poor survival in hepatocellular carcinoma, lung adenocarcinoma, and glioblastoma. We demonstrate that the miRNA is released by an as-yet unidentified proteinaceous  miRNA release factor from the grasp of Argonaute before its degradation. Our analyses of the patient-derived transcriptomics data also show that XRN2, via its regulation of let-7, affects multiple pathways in a consistent manner across epithelial and glial cell lineages, and thus, is of critical pathophysiological significance. "Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.