ISL1 is necessary for auditory neuron development and tonotopic organization A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central compensatory plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.
Charting the human amygdala development across childhood and adolescence: Manual and automatic segmentation The developmental pattern of the amygdala throughout childhood and adolescence has been inconsistently reported in previous neuroimaging studies. Given the relatively small size of the amygdala on full brain MRI scans, discrepancies may be partly due to methodological differences in amygdalar segmentation. To investigate the impact of volume extraction methods on amygdala volume, we compared FreeSurfer, FSL and volBrain segmentation measurements with those obtained by manual tracing. The manual tracing method, which we used as the  gold standard, exhibited almost perfect intra- and inter-rater reliability. We observed systematic differences in amygdala volumes between automatic (FreeSurfer and volBrain) and manual methods. Specifically, compared with the manual tracing, FreeSurfer estimated larger amygdalae, and volBrain produced smaller amygdalae while FSL demonstrated a mixed pattern. The tracing bias was not uniform, but higher for smaller amygdalae. We further modeled amygdalar growth curves using accelerated longitudinal cohort data from the Chinese Color Nest Project. Trajectory modeling and statistical assessments of the manually traced amygdalae revealed linearly increasing and parallel developmental patterns for both girls and boys, although the amygdalae of boys were larger than those of girls. Compared to these trajectories, the shapes of developmental curves were similar when using the volBrain derived volumes. FreeSurfer derived trajectories had more nonlinearities and appeared flatter. FSL derived trajectories demonstrated an inverted U shape and were significantly different from those derived from manual tracing method. The use of amygdala volumes adjusted for total gray-matter volumes, but not intracranial volumes, resolved the shape discrepancies and led to reproducible growth curves curves between manual tracing and the automatic methods (except FSL). Our findings revealed steady growth of the human amygdala, mirroring its functional development across the school age. Methodological improvements are warranted for current automatic tools to achieve more accurate tracing of the amygdala at school age, calling for next generation tools.
A signalling axis involving CNOT3, Aurora B and ERK promotes differentiation and survival of mesendodermal progenitor cells Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B, which requires sequences in the NOT box domain of CNOT3, and regulates MAPK/ERK signalling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse ES cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of ectoderm. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4 and Wnt3. The double mutation affects interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2, impacting on survival of the differentiated ME cells. Our results identify an adaptor function for CNOT3 that regulates a key pathway in embryogenesis and cancer. 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. Groping in the fog: soaring migrants exhibit wider scatter in flight directions and respond differently to wind under low visibility conditions Atmospheric conditions are known to affect flight propensity, behaviour during flight, and migration route in birds. Yet, the effects of fog have only rarely been studied although they could disrupt orientation and hamper flight. Fog could limit the visibility of migrating birds such that they might not be able to detect landmarks that guide them during their journey. Soaring migrants modulate their flight speed and direction in relation to the wind vector to optimize the cost of transport. Consequently, landmark-based orientation, as well as adjustments of flight speed and direction in relation to wind conditions, could be jeopardized when flying in fog. Using a radar system operated in a migration bottleneck (Strait of Messina, Italy), we studied the behaviour of soaring birds under variable wind and fog conditions over two consecutive springs (2016 and 2017), discovering that migrating birds exhibited a wider scatter of flight directions and responded differently to wind under fog conditions. Birds flying through fog deviated more from the mean migration direction and increased their speed with increasing crosswinds. In addition, airspeed and groundspeed increased in the direction of the crosswind, causing the individuals to drift laterally. Our findings represent the first quantitative empirical evidence of flight behaviour changes when birds migrate through fog and explain why low visibility conditions could risk their migration journey. MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1. Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1.This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo HM kinase of S6K1. We show that rapamycin resistant S6K1 truncation mutant {Delta}NH{Delta}CT, continues to display HM phosphorylation with selective sensitivity toward torin-1. We also show that HM phosphorylation of wildtype S6K1and {Delta}NH{Delta}CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis analysis highlights the involvement of a conserved 19 amino acid stretch of S6K1 in mediating mTORC2 dependent HM phosphorylation. We finally show that deletion of this amino acid stretch leads to complete loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data identifies mTORC2 as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complex 1 and 2 act in tandem to activate the enzyme. Functional buffering via cell-specific gene expression promotes tissue homeostasis and cancer robustness Functional buffering that ensures biological robustness is critical for maintaining tissue homeostasis, organismal survival, and evolution of novelty. However, the mechanism underlying functional buffering, particularly in multicellular organisms, remains largely elusive. Here, we developed an inference index (C-score) for Cell-specific Expression- BUffering (CEBU), whereby functional buffering is mediated via expression of buffering genes in specific cells and tissues in humans. By computing C-scores across 684 human cell lines using genome-wide CRISPR screens and transcriptomic RNA-seq, we report that C- score-identified putative buffering gene pairs are enriched for members of the same duplicated gene family, pathway, and protein complex. Furthermore, CEBU is especially prevalent in tissues of low regenerative capacity (e.g., bone and neuronal tissues) and is weakest in highly regenerative blood cells, linking functional buffering to tissue regeneration. Clinically, the buffering capacity enabled by CEBU can help predict patient survival for multiple cancers. Our results reveal CEBU as a buffering mechanism contributing to tissue homeostasis and cancer robustness in humans.

Summary blurbWe unveil a genome-wide functional buffering mechanism, termed Cell-specific Expression Buffering (CEBU), whereby gene expression contributes to functional buffering in specific cell types and tissues. We link CEBU to genetic interactions, tissue homeostasis and cancer robustness. Living with high potassium: how plants balance nutrient acquisition during K-induced salt stress O_LIHigh potassium (K) in the growth medium is more toxic to plants than Na at similar concentrations. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown.
C_LIO_LIWe examined Arabidopsis thaliana and its extremophyte relative Schrenkiella parvula, using a comparative multi-omics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damage while sustaining growth.
C_LIO_LIA. thaliana showed limited capacity to curb excess K accumulation and prevent nutrient depletion contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. Facilitated by a targeted transcriptomic response, promoting nitrogen uptake along with other key nutrients and uninterrupted N assimilation into primary metabolites during excess K-stress allowed S. parvula to boost its antioxidant and osmolyte pools concurrently leading to sustained growth. Antithetically, A. thaliana showed transcriptional responses indicative of a poor balance between stress signaling, increased ROS levels, and reduced photosynthesis, subsequently leading to inhibited growth.
C_LIO_LIThe ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid non-specific stress signaling are two main deterministic steps towards building stress resilience to excess K+-induced salt stress.
C_LI Genomic hallmarks and therapeutic implications of cancer cell quiescence Therapy resistance in cancer is often driven by a subpopulation of cells that are temporarily arrested in a non-proliferative, quiescent state, which is difficult to capture and whose mutational drivers remain largely unknown. We developed methodology to uniquely identify this state from transcriptomic signals and characterised its prevalence and genomic constraints in solid primary tumours. We show quiescence preferentially emerges in the context of more stable, less mutated genomes which maintain TP53 integrity and lack the hallmarks of DNA damage repair deficiency, while presenting increased APOBEC mutagenesis. We employ machine learning to uncover novel genomic dependencies of this process, and validate the role of the centrosomal gene CEP89 as a modulator of proliferation/quiescence capacity. Lastly, we demonstrate that quiescence underlies unfavourable responses to various therapies exploiting cell cycle, kinase signalling and epigenetic mechanisms in single cell data, and propose a signature of quiescence-linked therapeutic resistance to further study and clinically track this state.

STATEMENT OF SIGNIFICANCEWe developed a robust transcriptomic signature of cellular quiescence, and employed it to systematically characterise proliferation/quiescence decisions in solid primary cancers and the genomic events influencing them. We propose CEP89 as a novel target whose suppression increases quiescence. Our expression signature of quiescence could be employed to track resistance to multiple anti-cancer compounds in a drug-tolerant persister cell setting. Cooperation of two opposite flagella allows high-speed swimming and active turning in zoospores Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when these biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, characteristics of individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its pattern from sinusoidal waves to power and recovery strokes, similar to Chlamydomonass breaststroke, to reorient its body to a new direction. Our study is a fundamental step towards a better understanding of the spreading of plant pathogens motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated "run-and-tumble" motility class exhibited by peritrichous bacteria. NOMA: a high-throughput microchip for robust, sequential measurements of secretions from the same single-cells Despite recent advances in single-cell analysis technologies, lacking simple methods to keep the live single-cells traceable for longitudinal detection reliably poses a significant obstacle in single-cell secretion analysis. Here we reported the high-density NOMA (narrow-opening microwell array) microchip that realized the retention of[&ge;] 97% of trapped single cells in dedicated spatial locations during repetitive detection procedures, verified with both adherent and suspension cells by two researchers independently. We applied it in monitoring single-cell protein secretions sequentially from the same single cells, and we found the digital protein secretion patterns dominate the protein secretion. We also demonstrated the microchip for longitudinally tracking IL-8 and the CD81+EV secretions from the same single-cells over days, which revealed the presence of "super secretors" within the cell population be more persistent to secrete protein or extracellular vesicle for an extended period. The NOMA platform reported here is simple, robust, and easy to operate for tracking sequential measurements from the same single cells, representing a novel and informative tool to inspire new observations in biomedical research.

Table of Contents Graphic

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org.highwire.dtl.DTLVardef@aa69c7org.highwire.dtl.DTLVardef@6bc79org.highwire.dtl.DTLVardef@3fcbbforg.highwire.dtl.DTLVardef@597f4b_HPS_FORMAT_FIGEXP  M_FIG C_FIG The compound of baicalein, wogonin, and oroxylin-A inhibits EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy. Multiparametric Platform for Profiling Lipid Trafficking in Human Leukocytes: Application for Hypercholesterolemia Systematic insight into cellular dysfunctions can improve understanding of disease etiology, risk assessment and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validated this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrated lipid storage data into a novel readout, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlated positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explained hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking from a few mls of blood and is applicable to dissect metabolic disorders, such as hypercholesterolemia.

MotivationWe have limited information on how cellular lipid uptake and processing differ between individuals and influence the development of metabolic diseases, such as hypercholesterolemia. Available assays are labor intensive, require skilled personnel and are difficult to scale to higher throughput, making it challenging to obtain systematic functional cell-based data from individuals. To overcome this problem, we established a scalable automated analysis pipeline enabling reliable quantification of multiple cellular readouts, including lipid uptake, storage and mobilization, from different white blood cell populations. This approach provides new personalized insights into the cellular basis of hypercholesterolemia and obesity.

Graphical Abstract

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org.highwire.dtl.DTLVardef@9222c1org.highwire.dtl.DTLVardef@27e289org.highwire.dtl.DTLVardef@89ba82org.highwire.dtl.DTLVardef@33c9cd_HPS_FORMAT_FIGEXP  M_FIG C_FIG Cluster-specific gene markers enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations.

Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology.

Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536.

RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI 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. Parent-offspring inference in inbred populations Genealogical relationships are fundamental components of genetic studies. However, it is often challenging to infer correct and complete pedigrees even when genome-wide information is available. For example, inbreeding can obfuscate genetic differences between individuals, making it difficult to even distinguish first-degree relatives such as parent-offspring from full siblings. Similarly, genotyping errors can interfere with the detection of genetic similarity between parents and their offspring. Inbreeding is common in natural, domesticated, and experimental populations and genotyping of these populations often has more errors than in human datasets, so efficient methods for building pedigrees under these conditions are necessary. Here, we present a new method for parent-offspring inference in inbred pedigrees called SPORE (Specific Parent-Offspring Relationship Estimation). SPORE is vastly superior to existing pedigree-inference methods at detecting parent-offspring relationships, in particular when inbreeding is high or in the presence of genotyping errors, or both. SPORE therefore fills an important void in the arsenal of pedigree inference tools.

Author SummaryKnowing the genealogical relationships among individuals is critical for genetic analyses, such as for identifying the mutations that cause diseases or that contribute to valuable agricultural traits such as milk production. Although many tools exist for establishing pedigrees using genetic information, these tools fail when individuals are highly inbred, such as in domesticated animals, or in groups of people in which consanguineous matings are common. Furthermore, existing tools do not work well when genetic information has errors at levels observed in modern datasets. Here, we present a novel approach to solve these problems. Our method is significantly more accurate than existing tools and more tolerant of errors in the genetic data. We expect that our method, which is simple to use and computationally efficient, will be a useful tool in a diversity of settings, from the studies of human and natural populations, to agricultural and experimental settings. Robust and annotation-free analysis of alternative splicing using short-read scRNA-seq data Although alternative splicing is a fundamental and pervasive aspect of gene expression in higher eukaryotes, it is often omitted from single-cell studies due to quantification challenges inherent to commonly used short-read sequencing technologies. Here, we undertake the analysis of alternative splicing across numerous diverse murine cell types from two large-scale single-cell datasets--the Tabula Muris and BRAIN Initiative Cell Census Network--while accounting for understudied technical artifacts and unannotated isoforms. We find strong and general cell-type-specific alternative splicing, complementary to total gene expression but of similar discriminatory value, and identify a large volume of novel isoforms. We specifically highlight splicing variation across different cell types in primary motor cortex neurons, bone marrow B cells, and various epithelial cells; and show that the implicated transcripts include many genes which do not display total expression differences. To elucidate the regulation of alternative splicing, we build a custom predictive model based on splicing factor activity, recovering several known interactions while generating new hypotheses, including potential regulatory roles for novel alternative splicing events in critical genes including Khdrbs3 and Rbfox1. We make our results available using public interactive browsers to spur further exploration by the community. Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques Coronavirus disease 2019 (COVID-19), which is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, continues to threaten global public health. Developing a vaccine that only requires single immunization but provides long-term protection for the prevention and control of COVID-19 is important. Here, we developed an adeno-associated virus (AAV)-based vaccine expressing a stable receptor-binding domain (SRBD) protein. The vaccine requires only a single shot but provides effective neutralizing antibodies (NAbs) over 598 days in rhesus macaques (Macaca mulatta). Importantly, our results showed that the NAbs were kept in high level and long lasting against authentic wild-type SARS-CoV-2, Beta, Delta and Omicron variants using plaque reduction neutralization test. Of note, although we detected pre-existing AAV2/9 antibodies before immunization, the vaccine still induced high and effective NAbs against COVID-19 in rhesus macaques. AAV-SRBD immune serum also efficiently inhibited the binding of ACE2 with RBD in the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1/P.2 (Gamma), B.1.617.2 (Delta), B.1.617.1/3(Kappa), and C.37 (Lambda) variants. Thus, these data suggest that the vaccine has great potential to prevent the spread of SARS-CoV-2. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

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

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Host phenology regulates ecological and eco-evolutionary feedbacks underlying parasite-host demographic cycles Parasite-host interactions can result in periodic population dynamics when parasites over-exploit host populations. The timing of host seasonal activity, or host phenology, determines the frequency and demographic impact of parasite-host interactions which may govern if the parasite can sufficiently over-exploit their hosts to drive population cycles. We describe a mathematical model of a monocyclic, obligate-killer parasite system with seasonal host activity to investigate the consequences of host phenology on host-parasite dynamics. The results suggest that parasites can reach the densities necessary to destabilize host dynamics and drive cycling in only some phenological scenarios, such as environments with short seasons and synchronous host emergence. Further, only parasite lineages that are sufficiently adapted to phenological scenarios with short seasons and synchronous host emergence can achieve the densities necessary to over-exploit hosts and produce population cycles. Host-parasite cycles can also generate an eco-evolutionary feedback that slows parasite adaptation to the phenological environment as rare advantageous phenotypes are driven to extinction when introduced in phases of the cycle where host populations are small and parasite populations are large. The results demonstrate that seasonal environments can drive population cycling in a restricted set of phenological patterns and provides further evidence that the rate of adaptive evolution depends on underlying ecological dynamics. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. De-noising Spatial Transcriptomics Data Based on Position and Image Information Spatial transcriptomics (ST) technologies provide gene expression close to or even superior to single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, the expression data captured by ST technologies suffer from high noise levels, as a result of the shallow coverage in each sequencing unit. The extra experimental steps for preserving the spatial locations of sequencing could result in even more severe noises, compared to regular single-cell RNA-sequencing (scRNA-seq). Fortunately, such noises could be largely removed by leveraging information from the physical locations of sequencing, and the tissue and cellular organization reflected by corresponding pathology images. In this work, we demonstrated the extensive levels of noise in ST data. We developed a mathematical model, named Sprod, to impute accurate ST gene expression based on latent space and graph learning of matched location and imaging data. We comprehensively validated Sprod and demonstrated its advantages over prior methods for removing drop-outs in scRNA-seq data. We further showed that, after adequate imputation by Sprod, differential expression analyses, pseudotime analyses, and cell-to-cell interaction inferences yield significantly more informative results. Overall, we envision denoising by Sprod to become a key first step to empower ST technologies for biomedical discoveries and innovations. Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time The earliest geochemical indicators of microbes--and the enzymes that powered them--extend back almost 3.8 billion years on our planet. Paleobiologists often attempt to understand these indicators by assuming that the behaviors of modern microbes and enzymes are consistent (uniform) with those of their predecessors. A uniformitarian assumption (i.e., the idea that fundamental geobiological processes have occurred in much the same manner over Earth history) seems at odds with our understanding of the inherent variability of living systems. Here, we examine whether a uniformitarian assumption for an enzyme thought to generate carbon isotope indicators of biological activity, RuBisCO, can be corroborated by independently studying the history of changes recorded within RuBisCOs genetic sequences. Specifically, we resurrected a Precambrian-age, ancient RuBisCO by engineering its ancient DNA inside a modern cyanobacterium genome and measured the engineered organisms fitness and carbon-isotope-discrimination profile. The envelope of ancestral RuBisCO isotopic fractionation observed here indicates that uniformitarian assumptions may be warranted, but with important caveats. Our results suggest that further inquiries that link molecule-level evolutionary changes with planet-level geochemical conditions are needed to discern whether enzyme-affected isotope fractionation trends extend deeper into the early Precambrian. Experimental studies illuminating lifes early molecular innovations are crucial to explore the foundations of Precambrian uniformitarian assumptions. High Spatial Resolution MALDI Imaging Mass Spectrometry ofFresh-Frozen Bone Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracking, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 m) MALDI IMS was employed to characterize lipid distributions in fresh-frozen bone, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.