In vitro culture of bovine fibroblasts using select serum-free media supplemented with Chlorella vulgaris extract Standard cell culture practices require addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5-10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the cost of media while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC-MS, we compared molecular signatures of FBS to Chlorella algae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented with C. vulgaris extract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover, C. vulgaris extract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present in C. vulgaris extract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications.
NGF-p75 signaling coordinates skeletal cell migration during bone repair Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to re-innervation and osteogenesis is unknown. Here, we found that NGF is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to impact their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.
Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Utility of SARS-CoV-2 infection models using in vitro and in vivo ACE2-lentivirus transduction SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research. SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein Histidines Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing. Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation at many enhancers, distal regulatory elements that control spatio-temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific, code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high.

ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that is able to predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Then, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity and liver enhancers associated with liver regeneration have a stronger tendency than the general population to have predicted lineage-specific open chromatin.

ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements. EMBED: Essential Microbiome Dynamics, a dimensionality reduction approach for longitudinal microbiome studies Dimensionality reduction can offer unique insights into high dimensional microbiome dynamics by leveraging collective abundance fluctuations of multiple bacteria driven by similar ecological perturbations. However, methods providing lower-dimensional representations of microbiome dynamics both at the community and individual taxa level are not currently available. To that end, we present EMBED: Essential MicroBiomE Dynamics, a probabilistic non-linear tensor factorization approach. Similar to normal mode analysis in structural biophysics, EMBED infers ecological normal modes (ECNs), which represent the unique orthogonal modes capturing the collective behavior of microbial communities. A very small number of ECNs can accurately approximate microbiome dynamics across multiple data sets. Inferred ECNs reflect specific ecological behaviors, providing natural templates along which the dynamics of individual bacteria may be partitioned. Moreover, the multi-subject treatment in EMBED systematically identifies subject-specific and universal abundance dynamics that are not detected by traditional approaches. Collectively, these results highlight the utility of EMBED as a versatile dimensionality reduction tool for studies of microbiome dynamics. Human cells contain myriad excised linear introns with potential functions in gene regulation and as RNA biomarkers By using TGIRT-seq, we identified >8,500 short full-length excised linear intron (FLEXI) RNAs in human cells. Subsets of FLEXIs accumulated in a cell-type specific manner, and [~]200 corresponded to agotrons or mirtrons or encoded snoRNAs. Analysis of CLIP-seq datasets identified potential interactions between FLEXIs and >100 different RNA-binding proteins (RBPs), 53 of which had binding sites in [&ge;]30 different FLEXIs. In addition to proteins that function in RNA splicing, these 53 RBPs included transcription factors, chromatin remodeling proteins, and cellular growth regulators that impacted FLEXI host gene alternative splicing and/or mRNA levels in knockdown datasets. We computationally identified six groups of RBPs whose binding sites were enriched in different subsets of FLEXIs: AGO1-4 and DICER associated with agotrons and mirtrons; AATF, DKC1, NOLCI, and SMNDC1 associated with snoRNA-encoding FLEXIs; two different combinations of alternative splicing factors found in stress granules; and two novel RBP-intron combinations, one including LARP4 and PABC4, which function together in the cytoplasm to regulate ribosomal protein translation. Our results suggest a model in which proteins involved in transcriptional regulation, alternative splicing, or post-splicing secondary functions bind and stabilize cell-type specific subsets of FLEXIs that perform different biological functions and have potential utility as biomarkers. Simpati: patient classifier identifies signature pathways based on similarity networks for the disease prediction BACKGROUNDPathway-based patient classification is a supervised learning task which implies a model learning pathways as features to predict the classes of patients. The counterpart of enrichment tools for the pathway analysis are fundamental methods for clinicians and biomedical scientists. They allow to find signature cellular functions which help to define and annotate a disease phenotype. They provide results which lead human experts to manually classify patients. It is a paradox that pathwaybased classifiers which natively resolve this objective are not strongly developed. They could simulate the human way of thinking, decipher hidden multivariate relationships between the deregulated pathways and the disease phenotype, and provide more information than a probability value. Instead, there are currently only two classifiers of such kind, they require a nontrivial hyperparameter tuning, are difficult to interpret and lack in providing new insights. There is the need of new classifiers which can provide novel perspectives about pathways, be easy to apply with different biological omics and produce new data enabling a further analysis of the patients.

RESULTSWe propose Simpati, an innovative and interpretable patient classifier based on pathway-specific patient similarity networks. The first classifier to adopt ad-hoc novel algorithms for such graph type. It standardizes the biological high-throughput dataset of patients profiles with a propagation algorithm that considers the interconnected nature of the cells molecules for inferring a new activity score. This allows Simpati to classify with dense, sparse, and non-homogenous omic data. Simpati organizes patients molecules in pathways represented by patient similarity networks for being interpretable, handling missing data and preserving the patient privacy. A network represents patients as nodes and a novel similarity measure determines how much every pair act co-ordinately in a pathway. Simpati detects signature biological processes based on how much the topological properties of the related networks separate the patient classes. In this step, it includes a new cohesive subgroup detection algorithm to handle patients not showing the same pathway activity as the other class members. An unknown patient is then classified by a unique recommender system which considers how much is similar to known patients and distant from being an outlier. Simpati outperforms previously published classifiers on five cancer datasets described with two biological omics, classifies well with sparse data, identifies more relevant pathways associated to the patients disease than the competitors and has the lowest computational requirements.

CONCLUSIONSimpati can serve as generic-purpose pathway-based classifier of patient classes. It provides signature pathways to unveil the altered biological mechanisms of a disease phenotype and to classify patients according to the learnt pathway-specific similarities. The signature condition and patient prediction can be deciphered considering the patient similarity networks which must reveal the members of a patient class more cohesive and similar than the non-members. Simpati divides the pathways in up and downinvolved. Upinvolved when the signaling cascades generated by the altered molecules of the disease patients impact stronger the pathway than the ones of the control class. We provide an R implementation, a graphical user interface and a visualization function for the patient similarity networks. The software is available at: https://github.com/LucaGiudice/Simpati Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. Beetle bioluminescence outshines aerial predators Bioluminescence is found across the tree of life and has many functions. Yet we understand very little about its timing and origins, particularly as a predator avoidance strategy. Understanding the timing between bioluminescence and predator origins has yet to be examined and can help elucidate the evolution of this ecologically important aposematic signal. Using the most prevalent bioluminescent group, fireflies, where bioluminescence primarily functions as aposematic and sexual signals, the timing for the origins of both potential predators of fireflies and bioluminescence is explored. Divergence time estimations were performed using genomic-scale phylogenetic reconstructions, and multiple fossil calibration points, allowing for a robust estimate for the origin of lampyrid bioluminescence as a terrestrial and as an aerial signal. Our results recover the origins of terrestrial beetle bioluminescence at 141.17 (122.63-161.17) mya and firefly aerial bioluminescence at 133.18 (117.86-152.47) mya with a large dataset focused on Lampyridae; and terrestrial bioluminescence as 148.03 (130.12-166.80) mya, with the age of aerial bioluminescence at 104.97 (99.00-120.90) mya with a complementary broad Elateroidea dataset. These ages predate the origins of all known extant aerial predators (i.e., bats and birds) and support the much older terrestrial predators (assassin bugs, frogs, ground beetles, lizards, snakes, hunting spiders, and harvestmen) as the most likely drivers of terrestrial bioluminescence in beetles, and sexual signaling likely being the original function in aerial fireflies. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. DNA Strand Breaks and Gaps Direct Retroviral Integration Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration. Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8. Source-sink connectivity: A novel interictal EEG marker for seizure localization Over 15 million epilepsy patients worldwide have drug-resistant epilepsy (DRE). Successful surgery is a standard of care treatment for DRE but can only be achieved through complete resection or disconnection of the epileptogenic zone (EZ), the brain region(s) where seizures originate. Surgical success rates vary between 20-80% because no clinically validated biological markers of the EZ exist. Localizing the EZ is a costly and time-consuming process beginning with non-invasive neuroimaging and often followed by days to weeks of intracranial EEG (iEEG) monitoring. Clinicians visually inspect iEEG data to identify abnormal activity (e.g., low-voltage high frequency activity) on individual channels occurring immediately before seizures or spikes that occur on interictal iEEG (i.e., between seizures). In the end, the clinical standard mainly relies on a small proportion of the iEEG data captured to assist in EZ localization (minutes of seizure data versus days of recordings), missing opportunities to leverage these largely ignored interictal data to better diagnose and treat patients.

Intracranial EEG offers a unique opportunity to observe epileptic cortical network dynamics but waiting for seizures increases patient risks associated with invasive monitoring. In this study, we aim to leverage interictal iEEG data by developing a new network-based interictal iEEG marker of the EZ. We hypothesize that when a patient is not clinically seizing, it is because the EZ is inhibited by other regions. We developed an algorithm that identifies two groups of nodes from the interictal iEEG network: those that are continuously inhibiting a set of neighboring nodes ("sources") and the inhibited nodes themselves ("sinks"). Specifically, patient-specific dynamical network models (DNMs) were estimated from minutes of iEEG and their connectivity properties revealed top sources and sinks in the network, with each node being quantified by source-sink metrics (SSMs). We validated the SSMs in a retrospective analysis of 65 patients by using the SSMs of the annotated EZ to predict surgical outcomes. The SSMs predicted outcomes with an accuracy of 79% compared to an accuracy of 43% for clinicians predictions (surgical success rate of this dataset). In failed outcomes, we identified regions of the brain with high SSMs that were untreated. When compared to high frequency oscillations, the most commonly proposed interictal iEEG feature for EZ localization, SSMs outperformed in predictive power (by a factor of 1.2) suggesting SSMs may be an interictal iEEG fingerprint of the EZ. 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. Adapting to extremes: reconstructing evolution in response to changing climate over time and space in the diverse Australian plant genus Acacia AimMacroevolutionary analysis is increasingly being used to study biodiversity responses to climate change, for example by using phylogenetic node ages to infer periods of diversification, or phylogenetic reconstruction of traits to infer adaptation to particular stresses. Here we apply a recently developed macroevolutionary method to investigate the responses of a diverse plant genus, Acacia, to increasing aridity and salinity in Australia from the Miocene to the present. We ask whether increase in tolerance of aridity and salinity coincided with periods of aridification, and if it allowed the radiation of Acacia into a wide range of niches.

TaxonAcacia

LocationAustralia

MethodsWe applied the Niche Evolution Model (NEMo), which combines Environmental (or Ecological) Niche Modelling (ENM) with phylogenetic comparative methods (PCM) in a single statistical framework, to a large database of Acacia presence-only records and presence-absence survey sites in order to infer current environmental tolerances of Australia Acacia species and reconstruct the evolution of environmental tolerance to increasing aridity and salinity.

ResultsWe find that patterns in evolution of Acacia, over time and across different habitat types, are consistent with the aridification history of Australia and suggests substantial ability to adapt to high aridity and salinity.

Main conclusionsOur results suggest that many Acacia lineages have been able to exploit new environments created during the aridification of Australia through evolution of environmental tolerance, resulting in their current dominance of many habitats across the continent. This study demonstrates that phylogenetic studies of the evolution of responses to changing environment can move beyond application of simple trait-based models, allowing the underlying processes of speciation, adaptation and dispersal to be explicitly modelled in a macroecological and macroevolutionary context.

Statement of significanceAcacia species are found throughout Australia, from rainforests to deserts, and are striking in their environmental adaptability, so they are a perfect case study for understanding evolution of tolerance to environmental extremes in a changing climate. We use the largest database of spatial distribution records yet assembled, using both surveys and atlas data, and a new analytical method that combines the strengths of environmental niche modelling with phylogenetic comparative methods, to demonstrate rapid evolution in aridity and salinity tolerance in response to aridification of the Australian continent during the Neogene and Quaternary. Land use change through the lens of macroecology: insights from Azorean arthropods and the Maximum Entropy Theory of Ecology Human activity and land management practices, in particular land use change, have resulted in the global loss of biodiversity. These types of disturbance affect the shape of macroecological patterns, and therefore analyzing these patterns can provide insights into how ecosystems are affected by land use change. We here use arthropod census data from 96 sites at Terceira Island in the Azores archipelago across four different land uses of increasing management intensity: native forest, exotic forest, semi-natural pasture, and intensive pasture, to examine the effects of land use type on three macroecological patterns: the species abundance distribution, the metabolic rate distribution of individuals, and the species-area relationship. The Maximum Entropy Theory of Ecology (METE) has successfully predicted these patterns across habitats and taxa in undisturbed ecosystems, and thus provides a null expectation for their shapes. Across these patterns, we find that the forest habitats are the best fit by METE predictions, while the semi-natural pasture is consistently the worst fit, and the intensive pasture is intermediately well fit. We show that the direction of failure of the METE predictions at the pasture sites is likely due to the hyper-dominance of introduced spider species present there. We hypothesize that the particularly poor fit for the semi-natural pasture is due to the mix of arthropod communities out of equilibrium, leading to greater heterogeneity in composition and complex dynamics that violate METEs assumption of static state variables. The comparative better fit for the intensive pasture plausibly results from more homogeneous arthropod communities that are well adapted to intensive management, and thus whose state variables are less in flux. Analyzing deviations from theoretical predictions across land use type provides useful information about how land use and disturbance affect ecosystems, and such comparisons could be useful across other habitats and taxa. A new method based on the von Mises-Fisher distribution shows that a minority of liver-localized CD8 T cells display hard-to-detect attraction to Plasmodium-infected hepatocytes Malaria is a disease caused by Plasmodium parasites, resulting in over 200 million infections and 400,000 deaths every year. A critical step of malaria infection is when sporozoites, injected by mosquitoes, travel to the liver and form liver stages. Malaria vaccine candidates which induce large numbers of malaria-specific CD8 T cells in mice are able to eliminate all liver stages, preventing fulminant malaria. However, how CD8 T cells find all parasites in 48 hours of the liver stage lifespan is not well understood. Using intravital microscopy of murine livers, we generated unique data on T cell search for malaria liver stages within a few hours after infection. To detect attraction of T cells to an infection site, we used the von Mises-Fisher distribution in 3D, similar to the 2D von Mises distribution previously used in ecology. Our results suggest that the vast majority (70-95%) of malaria-specific and non-specific liver-localized CD8 T cells did not display attraction towards the infection site, suggesting that the search for malaria liver stages occurs randomly. However, a small fraction (15-20%) displayed weak but detectable attraction towards parasites which already had been surrounded by several T cells. We found that speeds and turning angles correlated with attraction, suggesting that understanding mechanisms that determine the speed of T cell movement in the liver may improve the efficacy of future T cell-based vaccines. Stochastic simulations suggest that a small movement bias towards the parasite dramatically reduces the number of CD8 T cells needed to eliminate all malaria liver stages, but to detect such attraction by individual cells requires data from long imaging experiments which are not currently feasible. Importantly, as far as we know this is the first demonstration of how activated/memory CD8 T cells might search for the pathogen in nonlymphoid tissues a few hours after infection. We have also established a framework for how attraction of individual T cells towards a location in 3D can be rigorously evaluated. Elucidation of SARS-CoV-2 budding mechanisms through molecular dynamics simulations of M and E protein complexes SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding. D1- and D2-like Receptors Differentially Mediate the Effects of Dopaminergic Transmission on Cost/Benefit Evaluation and Motivation in Monkeys It has been widely accepted that dopamine (DA) plays a major role in motivation, yet the specific contribution of DA signaling at D1-like receptor (D1R) and D2-like receptor (D2R) to cost-benefit trade-off remains unclear. Here, by combining pharmacological manipulation of DA receptors (DARs) and positron emission tomography imaging, we assessed the relationship between the degree of D1R/D2R blockade and changes in benefit- and cost-based motivation for goal-directed behavior of macaque monkeys. We found that the degree of blockade of either D1R or D2R was associated with a reduction of the positive impact of reward amount and increasing delay-discounting. Workload-discounting was selectively increased by D2R antagonism. In addition, blocking both D1R and D2R had a synergistic effect on delay-discounting but an antagonist effect on workload-discounting. These results provide fundamental in-sight into the distinct mechanisms of DA action in the regulation of the cost and benefit-based motivation, which have important implications for motivational alterations in both neurological and psychiatric disorders. 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. Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCECOVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis. Human papillomavirus integration transforms chromatin to drive oncogenesis Human papillomavirus (HPV) drives almost all cervical cancers and up to [~]70% of head and neck cancers. Frequent integration into the host genome occurs only for tumourigenic strains of HPV. We hypothesized that changes in the epigenome and transcriptome contribute to the tumourigenicity of HPV. We found that viral integration events often occurred along with changes in chromatin state and expression of genes near the integration site. We investigated whether introduction of new transcription factor binding sites due to HPV integration could invoke these changes. Some regions within the HPV genome, particularly the position of a conserved CTCF binding site, showed enriched chromatin accessibility signal. ChIP-seq revealed that the conserved CTCF binding site within the HPV genome bound CTCF in 4 HPV+ cancer cell lines. Significant changes in CTCF binding pattern and increases in chromatin accessibility occurred exclusively within 100 kbp of HPV integration sites. The chromatin changes co-occurred with out-sized changes in transcription and alternative splicing of local genes. We analyzed the essentiality of genes upregulated around HPV integration sites of The Cancer Genome Atlas (TCGA) HPV+ tumours. HPV integration upregulated genes which had significantly higher essentiality scores compared to randomly selected upregulated genes from the same tumours. Our results suggest that introduction of a new CTCF binding site due to HPV integration reorganizes chromatin and upregulates genes essential for tumour viability in some HPV+ tumours. These findings emphasize a newly recognized role of HPV integration in oncogenesis. Do males know? Evidence-driven rainmaking mating strategy by male bonobos to meet the fertile window of females Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success. An interplay between cellular growth and atypical fusion defines morphogenesis of a modular glial niche Neural stem cells (NSCs) are embedded in a multi-layered, intricate cellular microenvironment supporting their activity, the niche. Whilst shape and function are inseparable, the morphogenetic aspects of niche development are poorly understood. Here, we use the formation of the glial network of a NSC niche to investigate acquisition of architectural complexity. Cortex glia (CG) in Drosophila regulate neurogenesis and build a reticular structure around NSCs. We first show that individual CG cells grow tremendously to ensheath several NSC lineages, eventually spanning the entire tissue while partitioning the NSC population. Elaborate proliferative mechanisms convert these cells into syncytia rich in cytoplasmic bridges. Unexpectedly, CG syncytia further undergo homotypic cell-cell fusion, relying on defined molecular players of cell fusion such as cell surface receptors and actin regulators. Exchange of cellular components is however dynamic in space and time, a previously unreported unique mechanism. This atypical cell fusion remodels cellular borders, restructuring the CG syncytia. Ultimately, the coordination of growth and fusion builds the multi-level architecture of the niche, and creates a modular, spatial partition of the NSC population. Our findings provide novel insights into how a niche forms and organises while developing intimate contacts with a stem cell population.