Hot and cold spots of pest-induced US urban tree death, 2020-2050 O_LIUrban trees are important nature-based solutions for future wellbeing and livability but are at high risk of mortality from insect pests. In the United States (US), 82% of the population live in urban settings and this number is growing, making urban tree mortality a matter of concern for most of its population. Until now, the magnitudes and spatial distributions of risks were unknown.
C_LIO_LIHere, we combine new models of street tree populations in [~]30,000 US communities, species-specific spread predictions for 57 invasive insect species, and estimates of tree death due to insect exposure for 48 host tree genera.
C_LIO_LIWe estimate that 1.4 million street trees will be killed by invasive insects from 2020 through 2050, costing an annualized average of US$ 30M. However, these estimates hide substantial variation: 23% of urban centers will experience 95% of all insect-induced mortality. Further, 90% of all mortality will be due to emerald ash borer (Agrilus planipennis, EAB), which is expected to kill virtually all ash trees (Fraxinus spp.) in >6000 communities.
C_LIO_LIWe define an EAB high-impact zone spanning 902,500km2, largely within the southern and central US, within which we predict the death of 98.8% of all ash trees. "Mortality hotspot cities" include Milwaukee, WI; Chicago, IL; and New York, NY.
C_LIO_LIWe identify Asian wood borers of maple and oak trees as the highest risk future invaders, where a new establishment could cost US$ 4.9B over 30 years.
C_LIO_LIPolicy implications: To plan effective mitigation, managers must know which tree species in which communities will be at the greatest risk, as well as the highest-risk insects. We provide the first country-wide, spatial forecast of urban tree mortality due to invasive insect pests. This framework identifies dominant pest insects and spatial impact hotspots, which can provide the basis for spatial prioritization of spread control efforts such as quarantines and biological control release sites. Our results highlight the need for EAB early-detection efforts as far from current infestations as Seattle, WA. Further, these findings produce a list of biotic and spatiotemporal risk factors for future high-impact US urban forest insect pests.
C_LI
Mycorrhizal fungi influence disease during epidemics in plant populations O_LIWhile pathogenic and mutualistic microbes are ubiquitous across ecosystems and often co-occur within hosts, how they interact to determine patterns of disease in genetically diverse host populations is unknown.
C_LIO_LITo test whether microbial mutualists provide protection against pathogens, and whether this is influenced by host genotype, we conducted a field experiment in three naturally-occurring epidemics of a fungal pathogen, Podosphaera plantaginis, infecting a host plant, Plantago lanceolata, in the [A]land Islands, Finland. In each population, we collected epidemiological data on experimental plants, originating from six allopatric populations, which had been inoculated with a mixture of mutualistic mycorrhizal fungi or a non-mycorrhizal control.
C_LIO_LIInoculation with mycorrhizal fungi introduced benefits to host from every population, such as increased host growth and reduced disease severity. Host genotypes that were more susceptible to the pathogen received stronger protective effects from mycorrhizal fungi. However, inoculation with mycorrhizal fungi also increased host infection risk.
C_LIO_LIOur results show that mycorrhizal fungi introduce growth and defensive benefits, and shift infection risks in host populations under pathogen attack. Understanding how mutualists alter host susceptibility to disease will be important for predicting infection outcomes in ecological communities and in agriculture.
C_LI

Plain Language SummaryBeneficial,  mycorrhizal fungi in roots help plants grow and can protect them from disease caused by other, pathogenic microbes. This study shows that mycorrhizal fungi influence patterns of plant disease during pathogen outbreaks in a natural landscape.
Risk modelling of single cell populations revealed the heterogeneity of immune infiltration in hepatocellular carcinoma Hepatocellular carcinoma (HCC) is one of the most common primary hepatic malignancies. E2F transcription factors play an important role in the tumorigenesis and progression of HCC, mainly through the RB/E2F pathway. Prognostic models for HCC based on gene signatures have been developed rapidly in recent years; however, their discriminating ability at the single cell level remains elusive, which could reflect the underlying mechanisms driving the sample bifurcation. In this study, we constructed and validated a predictive model based on E2F expression, successfully stratifying HCC patients into two groups with different survival risks. Then we used a single-cell dataset to test the discriminating ability of the predictive model on infiltrating T cells, demonstrating remarkable cellular heterogeneity as well as altered cell fates. We identified distinct cell subpopulations with diverse molecular characteristics. We also found that the distribution of cell subpopulations varied considerably across onset stages among patients, providing fundamental basis for patient-oriented precision evaluation. Moreover, single-sample gene set enrichment analysis revealed that subsets of CD8+ T cells with significantly different cell adhesion level could be associated with different patterns of tumor cell dissemination. Therefore, our findings linked the conventional prognostic gene signature to the immune microenvironment and cellular heterogeneity at the single-cell level, thereby providing deeper insights into the understanding of HCC tumorigenesis. Mouse lemur, a new animal model to investigate cardiac pacemaker activity in primates. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Algal photosystem I dimer and high resolution model of PSI:plastocyanin complex Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here, we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Our cryo-EM structure identifies that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-LHCI monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH, and thus partially overlaps with the surface area that would bind one of the LHCII complexes in state transitions. We also define the most accurate available PSI-LHCI model at 2.3 [A] resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations of all the pigments, as well as 621 water molecules that affect energy transfer pathways. Principles of mRNA targeting via the Arabidopsism6A-binding protein ECT2 Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins. Genome-wide association study suggests an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. Additionally, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels. Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age. Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse (Slc:ICR) embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the forty chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Impact StatementLow-toxic super-resolution observation enables chromosome counting in preimplantation embryos without cell collection. Wolbachia load variation in Drosophila is more likely caused by drift than by host genetic factors Symbiosis is a continuum of long-term interactions ranging from mutualism to parasitism, according to the balance between costs and benefits for the protagonists. The density of endosymbionts is, in both cases, a key factor that determines both the transmission of symbionts and the host extended phenotype, and is thus tightly regulated within hosts. However, the evolutionary and molecular mechanisms underlying bacterial density regulation are currently poorly understood. In this context, the symbiosis between the fruit fly and its intracellular bacteria Wolbachia (wMelPop strain) is particularly interesting to study. Although vertically transmitted, the symbiont is pathogenic, and a positive correlation between virulence and wMelPop density is observed. In addition, the number of repeats of a bacterial genomic region -Octomom-is positively correlated with Wolbachia density, underlying a potential genetic mechanism that controls bacterial density. Interestingly, the number of repeats varies between host individuals, but most likely also within them. Such genetic heterogeneity within the host could promote conflicts between bacteria themselves and with the host, notably by increasing within-host competition between symbiont genotypes through a process analogous to the tragedy of the commons. To characterize the determinisms at play in the regulation of bacterial density, we first introgressed wMelPop in different genetic backgrounds of D. melanogaster, and found different density levels and Octomom copy numbers in each host lineage. To determine whether such variations reflect a host genetic determinism on density regulation through Octomom copy number selection, we replicated the introgressions and performed reciprocal crosses on the two Drosophila populations with the most extreme density levels. In both experiments, we detected an absence of directionality in the patterns of infection, associated with a strong instability of these patterns across generations. Given that bacterial density was highly correlated with Octomom copy numbers in all experiments, these results rather suggest a strong influence of drift and a random increase in the frequency of certain bacterial variants. We then discuss how drift, both on the symbiont population during transmission and on the host population, could limit the efficiency of selection in such a symbiotic system, and the consequences of drift on the regulation of density and composition of bacterial populations. Immunomodulatory Contribution of Mast Cells to the Regenerative Biomaterial Microenvironment Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials. A variable-gain stochastic pooling motif mediates information transfer from receptor assemblies into NF-κB A myriad of inflammatory cytokines regulate signaling pathways to maintain cellular homeostasis. The IKK complex is an integration hub for cytokines that govern NF-{kappa}B signaling. In response to inflammation, IKK is activated through recruitment to receptor-associated protein assemblies. How and what information IKK complexes transmit about the milieu are open questions. Here we track dynamics of IKK complexes and nuclear NF-{kappa}B to identify upstream signaling features that determine same-cell responses. Experiments and modeling of single complexes reveals their size, number, and timing relays cytokine-specific information with feedback control that is independent of transcription. Our results provide evidence for variable-gain stochastic pooling, a noise-reducing motif that enables parsimonious and cytokine-specific information transfer. We propose that emergent properties of stochastic pooling are general principles of receptor signaling.

One Sentence SummaryA variable-gain stochastic pooling motif mediates robust and tunable information transmission from the extracellular milieu into the cell. SPIKE-TRIGGERED INTRASPINAL MICROSTIMULATION IMPROVES MOTOR PERFORMANCE IN AN AMBULATORY RAT MODEL OF SPINAL CORD INJURY The purpose of this study was to determine if spike-triggered intraspinal microstimulation (ISMS) results in improved motor performance in an ambulatory rat model of spinal cord injury (SCI). Experiments were carried out in adult male Sprague Dawley rats with 175 kdyn moderate T8 contusion injury. Rats were randomly assigned to one of two groups: Control or Activity Dependent Stimulation (ADS) therapy. Four weeks post-SCI, all rats were implanted with a recording electrode in the left hindlimb motor cortex and a fine-wire, custom-made stimulating electrode in the contralateral lumbar spinal cord. Intracortical and intraspinal microstimulation were used to find sites of similar hip representation areas, which were paired together for ADS therapy. In the ADS therapy group, spike-stimulus conditioning was administered for 4 hours/day, 4 days/week, for 4 weeks via a tethered cable in a testing chamber. During therapy sessions, single-unit spikes were discriminated in real time in the hindlimb motor cortex and used to trigger stimulation in the spinal cord ventral horn. The optimal stimulus intensity (50% ISMS movement threshold) and spike-stimulus delay (10ms) determined in preliminary anesthetized preparations were used during ADS. Control rats were similarly implanted with electrodes but did not receive stimulation therapy. Motor performances of each rat were evaluated before SCI contusion, once a week post-SCI for four weeks (prior to electrode implantation), and once a week post-conditioning for four weeks. Behavioral testing included BBB scoring, Ledged Beam walking, Horizontal Ladder walking, treadmill kinematics via the DigiGait and TreadScan system, and open field walking using OptiTrack kinematic analysis. BBB scores were significantly improved in ADS rats compared to Control rats after 1 week of therapy. In the ADS therapy rats, BBB scores were significantly improved after two weeks of ADS therapy when compared to pre-therapy. Foot fault scores on the Horizontal Ladder were significantly lower in ADS rats compared to pre-therapy ADS and Control rats after 1 week of therapy and returned to pre-injury measures after three weeks of ADS therapy. The Ledged Beam test and kinematic analysis using the DigiGait and TreadScan system showed deficits after SCI in both ADS and Control rats but there were no significant differences between groups after 4 weeks of ADS therapy. These results show that activity dependent stimulation after spinal cord injury using spike-triggered ISMS enhances behavioral recovery of locomotor function as measured by the BBB score and the Horizontal Ladder task. Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Serum N- and O-Glycopeptide Data Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics. Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. 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. Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of [~]107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of [~]106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts. Evaluation of methods for estimating coalescence times using ancestral recombination graphs The ancestral recombination graph (ARG) is a structure that describes the joint genealogies of sampled DNA sequences along the genome. Recent computational methods have made impressive progress towards scalably estimating whole-genome genealogies. In addition to inferring the ARG, some of these methods can also provide ARGs sampled from a defined posterior distribution. Obtaining good samples of ARGs is crucial for quantifying statistical uncertainty and for estimating population genetic parameters such as effective population size, mutation rate, and allele age. Here, we use standard neutral coalescent simulations to benchmark the estimates of pairwise coalescence times from three popular ARG inference programs: ARGweaver, Relate, and tsinfer+tsdate. We compare 1) the true coalescence times to the inferred times at each locus; 2) the distribution of coalescence times across all loci to the expected exponential distribution; 3) whether the sampled coalescence times have the properties expected of a valid posterior distribution. We find that inferred coalescence times at each locus are most accurate in ARGweaver, and often more accurate in Relate than in tsinfer+tsdate. However, all three methods tend to overestimate small coalescence times and underestimate large ones. Lastly, the posterior distribution of ARGweaver is closer to the expected posterior distribution than Relates, but this higher accuracy comes at a substantial trade-off in scalability. The best choice of method will depend on the number and length of input sequences and on the goal of downstream analyses, and we provide guidelines for the best practices. Correlation between speed and turning naturally arises for sparsely sampled cell movements Mechanisms regulating cell movement are not fully understood. One feature of cell movement that determines how far cells displace from an initial position is persistence, the ability to perform movements in a direction similar to the previous movement direction. Persistence is thus determined by turning angles between two sequential displacements and be characterized by an average turning angle or persistence time. Recent studies found that a cells average speed and turning are negatively correlated, suggesting a fundamental cell-intrinsic program whereby cells with a lower turning ability (i.e., larger persistence time) are intrinsically faster (or faster cells turn less). By simulating correlated or persistent random walks (PRWs) using two different frameworks (one based on von Mises-Fisher (vMF) distribution and another based on Ornstein-Uhlenbeck (OU) process) we show that the negative correlation between speed and turning naturally arises when cell trajectories are sub-sampled, i.e., when the frequency of sampling is lower than frequency at which cells make movements. This effect is strongest when the sampling frequency is on the order of magnitude with the typical cell persistence time and when cells vary in persistence time. Both conditions are observed for datasets of T cell movements in vivo that we have analyzed. In simulations the correlation arises due to randomness of cell movements resulting in highly variable persistence times for individual cells that with sub-sampling leads to large variability of average cell speeds. Interestingly, previously suggested methodology of calculating displacement of cell cohorts with different speeds resulted in similar results whether or not there is a cell-intrinsic correlation between cell speed and persistence. For both vMF- and OU-based simulations of PRWs we could find parameter values (distribution of persistence times, speeds, and sampling frequency) that matched experimentally measured correlations between speed and turning for two datasets of T cell movement in vivo suggesting that such simple correlations are not fully informative on the intrinsic link between speed and persistence. Our results thus suggest that sub-sampling may contribute to (and perhaps fully explains) the observed correlation between speed and turning at least for some cell trajectory data and emphasize the role of sampling frequency in inference of critical cellular parameters of cell motility such as speeds.

Secondary AbstractMeasurement of cell movements often results in a negative correlation between average speed and average turning angle suggesting an existence of a universal, cell-intrinsic movement program. We show that such a negative correlation may arise if cells in the population differ in their ability for persistent movement when the movement data are sub-sampled. We show that sub-sampling of cell trajectories generated using two different frameworks of persistent random walk can match the experimentally observed correlation between speed and turning for T cell movements in vivo. 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. Apolipoprotein M attenuates anthracycline cardiotoxicity and lysosomal injury ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity.

BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF).

MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls.

ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients. ApomTG mice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass. ApomTG attenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH.

ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy.

HighlightsO_LICirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathy
C_LIO_LIAnthracycline treatment reduces circulating ApoM in humans and mice
C_LIO_LIIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacy
C_LIO_LIAutophagic impairment is characteristic of human anthracycline cardiomyopathy
C_LI HOXB7 induces STAT3-mediated transformation and lung metastasis in an immortalized mammary gland NMuMG cells Genes from the homeobox family are often dysregulated in cancer. Particularly, homeobox B7 (HOXB7) amplification and overexpression correlate with poor prognosis in various cancers, such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, drug resistance, and angiogenesis. In this study, we show that HOXB7 overexpression correlates with a poor prognosis in patients with breast cancer. Coamplification of HOXB7 with ERBB2 enhanced the expression of ERBB2 and the malignancy of HER2-positive breast cancer cells. Additionally, HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line, NMuMG, induced cellular transformation through an activation of the JAK-STAT signaling pathway, which resulted in lung metastasis upon orthotropic transplantation in mice. These findings reveal that HOXB7 alone is sufficient to induce tumorigenesis and lung metastasis using HOXB7-expressing NMuMG cells. 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. Effect of Adolescent Stress on Adult Morphine-Induced Behavioral Sensitization is Dependent Upon Genetic Background 1Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence - a critical period of frontal lobe development - influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the prefrontal cortex compared to control mice. The specificity of the effects for C57BL/6J versus BALB/cJ mice provides evidence of a gene by environmental interaction influencing opioid behaviors. Long-term differences in stress reactivity or miRNA expression suggests two possible biological mechanisms to evaluate in future research. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. A dynamical systems treatment of transcriptomic trajectories in hematopoiesis Inspired by Waddingtons illustration of an epigenetic landscape, cell-fate transitions have been envisioned as bifurcating dynamical systems, wherein the dynamics of an exogenous signal couples to a cells enormously complex signaling and transcriptional machinery, eliciting a qualitative transition in the collective state of a cell - its fate. It remains unclear, however, whether the dynamical systems framework can go beyond a word-based caricature of the system and provide sharp quantitative insights that further our understanding of differentiation. Single-cell RNA sequencing (scRNA-seq), which measures the distributions of possible transcriptional states in large populations of differentiating cells, provides an alternate view, in which development is marked by the individual concentration variations of a myriad of genes. Here, starting from formal mathematical derivations, we challenge these transcriptomic trajectories to a rigorous statistical evaluation of whether they display signatures consistent with bifurcations. After pinpointing bifurcations along transcriptomic trajectories of the neutrophil branch of hematopoeitic differentiation we are able to further leverage the primitive features of a linear instability to identify the single-direction in gene expression space along which the bifurcation unfolds and identify possible gene contributors. This scheme identifies transcription factors long viewed to play a crucial role in the process of neutrophil differentiation in addition to identifying a host of other novel genetic players. Most broadly speaking, we provide evidence that, though very high-dimensional, a bifurcating dynamical systems formalism might be appropriate for the process of cellular differentiation and that it can be leveraged to provide insights. Ambitiously, our work attempts to take a step beyond data-analysis and towards the construction of falsifiable mathematical models that describe the dynamics of the entire transcriptome.